KR20170117107A - A fusion protein comprising a ligand binding domain of VEGF and PDGF - Google Patents

Abstract

A PDGF binding site, a VEGF binding site and an Fc antibody region. Also disclosed are methods of using fusion proteins to treat or prevent clinical symptoms characterized by abnormal nucleic acid, a composition comprising a fusion protein, and abnormal angiogenesis, such as vascular permeability, edema or inflammation.

Description

A fusion protein comprising a ligand binding domain of VEGF and PDGF

This application claims the benefit of US Provisional Application No. 62 / 131,261, filed March 11, 2015, at 35 U.S.C. The benefit of §119 (e) is claimed, the description of which is incorporated herein by reference.

The present application contains a sequence listing in ASCII format, filed March 3, 2016, electronically submitted via EFS-Web and having the file title "688947-3WO_ST25 ", which is approximately 88.9 kilobytes in capacity. Sequence listings are filed on EFS-web as part of the specification, which is incorporated by reference herein in its entirety.

The present invention relates to a composition comprising a PDGF binding portion, a VEGF binding portion, and an Fc antibody region, a nucleic acid encoding a fusion protein, and an expression vector, a fusion protein comprising the recombinant cells thereof, and a fusion protein. Methods of using fusion proteins to inhibit PDGF and VEGF function are also provided.

Angiogenesis is the formation of new blood vessels from existing blood vessels and is a normal and important process associated with fetal development and tissue repair. Angiogenesis is highly regulated by both angiogenic factors and antiangiogenic factors, including endothelial cell migration and proliferation, vascular maturation and remodeling, and degradation of extracellular matrix. Although angiogenesis is an important process for normal growth and development, it also plays a key role in tumor growth, ischemia and inflammation.

During acute uncontrolled ocular angiogenesis, increased vascular permeability leads to vascular vulnerability and leakage leading to accumulation of hemorrhage, body fluids, and protein exudates, ultimately resulting in vascular dysfunction or vascular hyperplasia. Ocular neovascularization can occur in the spectrum of visual impairments such as senile AMD, diabetic neuropathy (PDR), and corneal neovascularization. Both AMD and PDR can impair the structure and function of retinal nerve cells, ultimately causing visual loss. Untreated, abnormal blood vessels can induce fibrotic scars resulting in irreversible damage to retinal function and eventually blindness (Zhang and Ma, Prog Retin Eye Res 2007 Jan; 26 (l): 1-37). Corneal angiogenesis similarly reduces the transparency of the cornea and causes visual loss.

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. The human VEGF family includes VEGF-A VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PIGF). In addition, several isoforms of VEGFA, VEGF-B and PIGF are produced by alternative RNA splicing (Sullivan and Brekken, MAbs. 2010 Mar-Apr, 2 (2): 165-75 ). VEGF-A is a prototypic member of the family and is best characterized. VEGF-A acts as a mitogenic factor in endothelial cells, promotes endothelial cell survival and proliferation, induces cell migration, and increases microvascular permeability. The proteins of the VEGF family bind to the extracellular region of cell surface VEGF receptors (VEGFRs) to activate the VEGF signaling pathway and activate the VEGF signaling pathway.

There are three types of VEGFR proteins: VEGFR1, VEGFR2, and VEGFR3, which contain an extracellular region containing seven immunoglobulin (Ig) -like domains, respectively. The extracellular domain of VEGFRs binds to different VEGF proteins. For example, VEGFR-1 (Flt-1) binds to VEGF-A, VEGF-B and PIGF and can function as a regulator of VEGFs or a decoy receptor of VEGFR-2. VEGFR-2 (KDR / Flk-1) binds to all VEGF isoforms and is a major mediator of VEGF-induced angiogenic signaling. VEGFR-3 (Flt-4) binds to VEGF-C and VEGF-D, but not VEGF-A, and acts as a mediator of lymphangiogenesis.

The high molecular weight variants VEGF ₂₀₆ and VEGF _189K are tightly bound to the extracellular membrane and do not interact with the VEGF receptor. While VEGF ₁₆₅ is a predominant soluble variant, VEGF ₁₂₁ and VEGF _{145 are} also soluble variants that bind to the VEGFR1 and VEGFR2 receptors as well as the degradation product VEGF ₁₁₀ (Bhisitkuk, Br J Ophthalmol 2006 Dec; 90 (12): 1542 -7).

Blocking VEGF activity with inhibitors of antibodies, soluble VEGF receptors or VEGF tyrosine kinase activity is a strategy used to treat angiogenesis disorders such as AMD. Although anti-VEGF therapy generally stabilizes or enhances visual function, subretinal scar or fibrosis has been reported to occur within two years of treatment in approximately half of all treated eyes (Daniel et al., Ophthalmology. 2014 Mar; 121 (3): 656-66). In addition, targeting VEGF only prevents the formation of new blood vesicles, but does not affect newly formed blood vessels.

Recent data suggest that pericytes can play a role in anti-VEGF resistance, stabilization of new blood vessels and scarring. Pericyte cells interact with endothelial cells and contribute to the formation of blood-retinal barrier. In particular, peripheral vascular cells provide survival signals to vascular endothelial cells, making them resistant to VEGF reduction therapy (Benjamin et al, Development. 1998 May; 125 (9): 1591-8, Patel, Retina, 2009 Jun, 29 (6 Suppl) S45-8). Platelet derived growth factor (PDGF) regulates peripheral vascular cells to induce their recruitment, proliferation and survival and to regulate the maturation of new blood vessels.

The human PDGF family includes PDGF-A, PDGF-B, PDGF-C and PDGF-D. The four PDGF proteins form homologous or heterodimers such as PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, which are inactive in monomeric form. The dimeric protein binds to the extracellular domain of the cell surface PDGF receptor (PDGFR) and activates the PDGF signaling pathway.

There are two PDGF receptors, PDGFR-alpha and PDGFR-beta, which form homologous or heterodimers (e.g. PDGFR-alpha, PDGFR-beta beta and PDGFR- alpha beta) And an extracellular region. The ligand-binding site of the receptor is located in the first three Ig-like domains (D1 to D3).

The extracellular domain of PDGFR dimer binds to other PDGF proteins. For example, PDGFR-αa specifically interacts with PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC. PDGFR-αβ specifically interacts with PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD. PDGFR-ββ specifically interacts with PDGF-BB and PDGF-DD. PDGF-BB is the only PDGF capable of binding with high affinity to all three receptor dimer forms and is able to induce proliferation and migration of pericytes in vitro and in vivo . The extracellular domain consisting of five Ig-like domains of PDGFR-beta (D1 to D5) has been shown to antagonize previously stimulated responses by PDGF-B (Duan et al., J Biol Chem. 1991 Jan 5: 266 l): 413-8; Ueno et al., Science, 1991 May 10, 252 (5007): 844-8). Studies using PDGFRJ3-Fc chimeric proteins have demonstrated that D1 to D3 of human PDGFR-β is sufficient for high affinity PDGF-B ligand binding (Heidaran et al., FASEB J.1991; 9 (1): 140- 5, Lokker et al., J Biol Chem. 1997 Dec 26; 272 (52): 33037-44). In addition, preliminary dimerization of D1 to D3 of PDGFR-β fused to glutathione S-transferase (GST) improved binding affinity for PDGF-BB ligand compared to recombinant PDGFR-β D1-D3 protein (Leppanen et al., Biochemistry, 2000 Mar 7, 39 (9): 2370-5).

Current anti-VEGF therapies are very effective, but intensive patient monitoring and frequent treatment are required to achieve optimal results. In addition, the drug should be given indefinite treatment because it targets symptoms of the disease, not the underlying cause. Inadequate treatment can result in continued choroidal neovascularization (CNVs) that continue to grow and eventually mature into fibrotic scars resulting in irreversible visual loss (Martin et al., 119: 1388-1398; Bloch et al, Am J Ophthalmol. ; 156 (1): 16-124; Daniel et al., Ophthalmology, 2014 Mar; 121 (3): 656-66). Agents that can prevent neovascularization and induce immature angiogenesis in neovascular cortical lesions can eliminate the cause of vascular leakage and fibrosis, thereby reducing or eliminating the need for intensive patient monitoring and ongoing treatment.

Recently, fusion proteins including the extracellular portion of the PDGF receptor, the extracellular portion of the VEGF receptor, and the multimerization domain from the N-terminus to the C-terminus have been disclosed (US Publication No. 2014/0315804). The fusion protein binds both PDGF and VEGF and inhibits their activity.

Despite the advances described in the field of dual inhibitors of PDGF and VEGF, there is a need in the art for improved agents and therapies for angioplasty.

U.S. Publication No. 2014/0315804

Zhang and Ma, Prog Retin Eye Res 2007 Jan; 26 (1): 1-37 Sullivan and Brekken, MAbs. 2010 Mar-Apr, 2 (2): 165-75 Bhisitkuk, Br J Ophthalmol. 2006 Dec; 90 (12): 1542-7 Daniel et al, Ophthalmology. Mar 2014; 121 (3): 656-66 Benjamin et al, Development. 1998 May; 125 (9): 1591-8, Patel, Retina, 2009 Jun, 29 (6 Suppl) S45-8 Duan et al., J Biol Chem. 1991 Jan 5; 266 (1): 413-8; Ueno et al., Science. 1991 May 10, 252 (5007): 844-8 Heidaran et al., FASEB J.1991; 9 (1): 140-5 Lokker et al., J Biol Chem. 1997 Dec 26; 272 (52): 33037-44 Leppanen et al., Biochemistry, 2000 Mar 7, 39 (9): 2370-5 Martin et al., Ophthalmology 2012; 119: 1388-1398 Bloch et al, Am J Ophthalmol. Jul; 156 (1): 16-124 Daniel et al., Ophthalmology, 2014 Mar; 121 (3): 656-66

The present invention satisfies this need by providing a novel fusion protein that simultaneously binds both VEGF and PDGF and simultaneously targets both signal pathways. Fusion proteins were generated by fusing the extracellular ligand binding domain derived from VEGF and PDGF receptors to the half-life extended Fc domain of IgG1.

In one general aspect, the invention provides a kit comprising (a) a first peptide comprising an extracellular ligand binding domain of a VEGF receptor, (b) an Fc region of an antibody, and (c) an extracellular ligand binding domain of a PDGF receptor To a fusion protein comprising a second peptide; Wherein said fusion protein is arranged at the N-terminus to the C-terminus in an order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a); The fusion protein can bind to VEGF and PDGF and inhibit the activity of VEGF and the activity of PDGF.

In one embodiment of the invention, the extracellular ligand binding domain of the VEGF receptor is capable of binding to the VEGF ligand and comprises Ig-like domains D1-D7 of one or more VEGF receptors. Preferably, the extracellular ligand-binding domain of the VEGF receptor comprises an Ig-like domain D2 of the first VEGF receptor and an Ig-like domain D3 of the second VEGF receptor, wherein the first and second VEGF receptors are identical or different VEGF Lt; / RTI > In one embodiment, the extracellular ligand binding domain of the VEGF receptor comprises an Ig-like domain D2 of VEGFR1 and an Ig-like domain D3 of VEGFR2. In another embodiment, the extracellular ligand binding domain of a VEGF receptor comprises an amino acid sequence having 90% or more sequence homology with SEQ ID NO: 7. More preferably, the extracellular ligand binding domain of the VEGF receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 10.

In one embodiment of the invention, the extracellular ligand-binding domain of a PDGF receptor is capable of binding to a PDGF ligand and comprises at least one Ig-like domain D1-D5 of one or more PDGF receptors. Preferably, the extracellular ligand-binding domain of the PDGF receptor comprises an Ig-like domain D1-D3 of one or more PDGF receptors. In one embodiment, the extracellular ligand binding domain of the PDGF receptor comprises an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 2. More preferably, the extracellular ligand binding domain of the PDGF receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 5.

In one embodiment of the invention, the Fc region of the antibody comprises the CH2 and CH3 regions of IgG1. Preferably, the Fc region of the antibody comprises an amino acid sequence having 90% or more sequence homology with SEQ ID NO: 12. More preferably, the Fc region of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 15.

In a preferred embodiment of the invention the fusion protein comprises (a) an Fc region of an antibody comprising Ig-like domain D2 of VEGFRl and Ig-like domain D3 of VEGFR2, (b) CH2 of CH3 and CH3 of IgG1, and (c) Lt; RTI ID = 0.0 > D1-D3 < / RTI > of PDGFR; The fusion protein is arranged at the C-terminus at the N-terminus in the order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a) c) - (b) - (a).

In one embodiment of the invention, the fusion protein further comprises a linker peptide between the Fc region and the first or second peptide at the C-terminus of the fusion protein, and optionally the second or first N- And a second linker peptide between the peptide and the Fc region.

In one embodiment of the invention, the fusion protein further comprises a signal peptide operably linked to the N-terminus of the fusion protein.

In another general aspect, the invention relates to isolated nucleic acid molecules encoding a fusion protein of the invention.

In another general aspect, the invention relates to an expression vector comprising a nucleic acid molecule encoding a fusion protein of the invention.

In another general aspect, the invention relates to a recombinant host cell comprising a nucleic acid molecule encoding a fusion protein of the invention.

In another general aspect, the invention relates to a method for producing a fusion protein of the invention. The method comprises (1) culturing a host cell comprising a nucleic acid molecule encoding a fusion protein under conditions under which the fusion protein is produced; And (2) recovering the fusion protein produced by the host cell.

In another general aspect, the invention relates to a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutically acceptable carrier.

In another general aspect, the invention relates to a pharmaceutical composition comprising a nucleic acid molecule encoding a fusion protein of the invention and a pharmaceutically acceptable carrier.

In another general aspect, the invention relates to a method of reducing the activity of VEGFR and the activity of PDGFR. The method comprises administering an effective amount of a fusion protein of the invention to an individual in need thereof.

In another general aspect, the invention provides a method of treating or preventing a clinical condition selected from the group consisting of neovascularization, vascular permeability, edema and inflammation . The method comprises administering an effective amount of a fusion protein according to an embodiment of the present invention to an individual in need thereof.

In another general aspect, the invention provides a method of treating a clinical condition selected from the group consisting of choroidal neovascularization, wet age-related macular degeneration, and geographic atrophy Or prevention. The method comprises administering an effective amount of a fusion protein according to an embodiment of the present invention to an individual in need thereof.

In one embodiment of the invention, the fusion protein is administered as a separate protein or expression vector.

In one embodiment of the invention, the clinical symptoms are selected from the group consisting of brain edema, stroke, cancer, psoriasis, arthritis, asthma, generalized edema associated with burns, ascites and pleural effusion associated with tumors, inflammation or trauma, chronic airway inflammation, capillary leak syndrome, Inflammatory arthritis, osteoarthritis, atherosclerosis < RTI ID = 0.0 > psoriasis, < / RTI > osteoarthritis, And aged-related macular degeneration, proliferative and nonproliferative diabetic retinopathy, corneal neovascularization (corneal neovascularization) or ocular inflammation and / or ocular angiogenesis, including ocular neovascularization, eu neovascularization, rubeosis iridis, and neovascular glaucoma.

In yet another general aspect, the present invention relates to dimeric antagonists of PDGF and VEGF comprising a fusion protein of the invention.

In yet another general aspect, the present invention relates to a protein conjugate comprising a fusion protein of the invention bound to one or more ligands selected from the group consisting of PDGF-BB and VEGF-A.

Other aspects, features, and advantages of the present invention will become apparent from the following description, including the detailed description of the invention and its preferred embodiments and appended claims.

In certain embodiments of the invention, all components of the fusion protein are of human origin and are therefore expected to be useful as non-immunogenic therapeutic agents in humans. Fusion proteins can inhibit VEGF and PDGF-dependent cell growth in vitro and can reduce retinal efflux by VEGF in animal models.

There is increasing evidence that angiogenesis can occur in the absence of VEGF signaling and that peri-vascular cells supply anti-VEGF resistance by supplying endothelial cells that proliferate VEGF and other cell survival factors (Reinmuth et al., FASEB J 2001 May; 15 (7): 123 9-41). Pericyte origin has also been suggested for muscle fibers that injure tissue and tumors. The PDGF signaling pathway is responsible for surrounding cell recruitment, survival and maturation (Andrae et al., Genes Dev. 2008 May 15; 22 (10): 1276-3 12). Inhibition of PDGF receptor signaling by antibodies has been shown to improve the efficacy of anti-VEGF treatment in a multiple mouse model of ocular neovascularization (Jo et al., Am J Pathol 2006 Jun; 168 (6): 2036-53 ). Large-scale, two-phase clinical trials of anti-VEGF and anti-PDGF agents, E10030, showed better results than anti-VEGF monotherapy (Dugel, Retina Today, March 2013, 65-71). Accordingly, the fusion protein of the present invention is effective for the treatment of angiogenic disorders such as AMD and cancer. A further advantage of the fusion protein is that there is no need to inject two separate compositions, an anti-PDGF agent and an anti-VEGF agent. Instead, a single composition comprising the fusion protein of both agents allows a single injection, thereby reducing the patient ' s risk of infection and injection trauma.

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It is to be understood that the invention is not limited to the precise embodiments shown in the drawings.
In the drawing:
Figure 1 depicts a bi-functional fusion protein designed to simultaneously inhibit both the PDGF and VEGF pathways according to embodiments of the present invention, showing fusion protein 2 (top) and fusion protein 1 (bottom) : PDGFR extracellular Ig-like domain (PID) represents extracellular Ig-like domains D1 to D3 of PDGFRP; Fc represents an IgGl CH2 and CH3 domain; The VEGFR extracellular Ig-like domain (VID) represents the extracellular Ig-like domain D2 of VEGFR1 and the extracellular Ig-like domain D3 of VEGFR2.
Figures 2a and 2b show SDS-PAGE gel analysis of purified i-functional Fc fusion proteins 1 and 2, respectively. Figure 2C shows SDS-PAGE gel analysis of reduced or non-reduced and purified i-functional Fc fusion proteins, respectively: lane M = marker; Lane 1 (non-reducing) and 2 (reducing) = positive control 2; Lane 3 (non-reducing) and 4 (reducing) = fusion protein 5; Lane 5 (non-reducing) and 6 (reducing) = fusion protein 3;
Figure 3 shows the results of a direct ligand binding assay of three test samples: purified positive control 1 (·), fusion protein 3 (A) and fusion protein 5 () for VEGF ₁₆₅ . The wells were precoated with VEGF ₁₆₅ and incubated with various concentrations of test samples; The amount of bound test sample was detected using FIRP-conjugated goat anti-human IgGl Fc specific antibody and OD ₄₅₀ readout plotted against test sample concentration;
Figure 4 shows the results of a direct ligand binding assay of three test samples: purified positive control 2 (·), fusion protein 3 (A) and fusion protein 5 () for PDGF-BB. The wells were precoated with PDGF-BB and incubated with various concentrations of test sample; The amount of bound test sample was detected using FIRP-conjugated goat anti-human IgGl Fc specific antibody and OD ₄₅₀ readout plotted against test sample concentration;
Figures 5a and 5b show the affinity evaluation of fusion protein 1 for VEGF ₁₆₅ and PDGF-BB in solution in competitive binding assays, respectively. Various concentrations of the fusion protein 1 were incubated overnight in solution with fixed concentrations of VEGF ₁₆₅ or PDGF-BB and the concentrations of free VEGF ₁₆₅ or PDGF-BB were measured using a quantitative sandwich ELISA assay and plotted against the concentration of fusion protein 1 Lt; / RTI >
Figures 6a and 6b show the affinity evaluation of fusion protein 2 for VEGF ₁₆₅ and PDGF-BB in solution in competitive binding assays, respectively. Various concentrations of fusion protein 2 were incubated overnight in solution with fixed concentrations of VEGF ₁₆₅ or PDGF-BB, and the concentration of free VEGF ₁₆₅ or PDGF-BB was measured using a quantitative sandwich ELISA assay and plotted against the concentration of fusion protein 2 Lt; / RTI >
FIG. 7 shows the inhibitory effect of positive control 1 (.), Fusion protein 3 (A) and fusion protein 5 () on the VEGF-dependent growth of HUVEC cells. The OD ₄₉₀ readings were plotted against the test sample concentration;
FIG. 8 shows the inhibitory effect of positive control 2 (.), Fusion protein 3 (A) and fusion protein 5 () on PDGF-dependent growth of BALB / 3T3 cells. The OD ₄₉₀ readings were plotted against the test sample concentration;

Various publications, articles and patents are cited or described throughout the background and specification. Each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like contained herein is provided to provide a background to the present invention. Such discussion does not admit that some or all of these matters constitute part of the prior art in connection with the invention disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meanings set forth in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein. It should be noted that the singular forms used in this specification and the appended claims include plural referents unless the context clearly dictates otherwise.

The present invention is directed to a composition comprising (a) a first peptide comprising an extracellular ligand binding domain of a VEGF receptor, (b) an Fc region of an antibody, and (c) a second peptide comprising an extracellular ligand binding domain of a PDGF receptor Lt; / RTI > fusion protein; Wherein said fusion protein is arranged at the N-terminus to the C-terminus in an order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a); The fusion protein may bind to VEGF-A and PDGF-BB and inhibit the activity of VEGFRl, VEFGFR2 and PDGFR.

Surprisingly, it has been found in the present invention that the orientation of the extracellular ligand binding domain of VEGF and PDGF receptors to other components in the fusion protein, such as the antibody Fc region, affects the binding affinity of the fusion protein for VEGF and PDGF ligands . Fusion proteins according to one embodiment of the present invention may have optimized affinity for both ligands and may have increased efficacy.

As used herein, the phrase "fusion protein" refers to a protein having two or more parts covalently linked, each portion of which is derived from a different protein.

As used herein, the term "VEGF" refers to any vascular endothelial growth factor protein that modulates the VEGF signaling pathway. Thus, the term VEGF may refer to VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, P1GF or isoforms thereof.

Refers to any receptor that binds to the terms "VEGF receptor" and "VEGFR" dms VEGF ligands, as used herein. Thus, the term VEGF receptor may refer to VEGFRl, VEGFR2 or VEGFR3.

As used herein, the phrase "extracellular ligand binding domain" refers to any region of the receptor protein that is located outside the cell and capable of binding to its ligand.

Thus, the extracellular ligand-binding domain of a VEGF receptor present in a fusion protein of the invention can be derived from any VEGFR, including but not limited to VEGFRl, VEGFR2 and VEGFR3. The seven extracellular IgG-like domains of the VEGFR protein are numbered 1, 2, 3, 4, 5, 6 and 7 from the N-terminus to the C-terminus of the extracellular domain and D1, D2, D3, D4 , D5, D6 and D7. The extracellular ligand binding domain of VEGFR present in the fusion protein of the invention may comprise one or more of the seven IgG-like domains from the extracellular domain of one or more optional VEGFR proteins. For example, the extracellular ligand-binding domain of a VEGF receptor present in a fusion protein of the invention can be at least one of D1, D2, D3, D4, D5, D6 or D7 from one or more of VEGFR1, VEGFR2, or VEGFR3 .

In a preferred embodiment, the extracellular ligand binding domain of the VEGF receptor present in the fusion protein comprises an Ig-like domain D2 of VEGFR1 and an Ig-like domain D3 of VEGFR2. Preferably, the extracellular ligand binding domain of VEGFR comprises one or more mutations that increase its binding to VEGF. In a more preferred embodiment, the extracellular ligand binding domain of the VEGF receptor present in the fusion protein comprises an amino acid sequence having 90% or more homology with SEQ ID NO: 7. In a more preferred embodiment, the extracellular ligand binding domain of the VEGF receptor present in the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 10.

The extracellular ligand-binding domain of the VEGF receptor present in the fusion protein of the present invention may be derived from any animal, such as a human or other suitable mammal such as a mouse, rabbit, rat, pig, dog or primate. In a preferred embodiment, the VEGFR is derived from a human.

As used herein, the term "PDGF" refers to any plasma-derived growth factor protein that modulates the PDGF signaling pathway. Thus, the term PDGF may refer to PDGF-A, PDGF-B, PDGF-C or PDGF-D.

As used herein, the terms "PDGF receptor" and "PDGFR" refer to any receptor that binds to a PDGF ligand.

The extracellular ligand binding domain of the PDGF receptor present in the fusion protein of the present invention may be derived from any PDGFR, including, but not limited to, PDGFR-a and PDGFR-beta. The five extracellular IgG-like domains of the PDGFR protein are numbered 1, 2, 3, 4 and 5 from the N-terminus to the C-terminus of the extracellular domain and are also referred to as D1, D2, D3, D4 and D5 . The extracellular ligand-binding domain of PDGFR present in the fusion protein of the invention may comprise one or more of the five IgG-like domains from the extracellular domain of one or more optional PDGFR proteins. For example, the extracellular ligand binding domain of a PDGF receptor present in a fusion protein of the invention can be at least one of D1, D2, D3, D4 or D5 from one or more of PDGFR-a or PDGFR-beta.

In a preferred embodiment, the extracellular ligand binding domain of the PDGF receptor present in the fusion protein comprises Ig-like domains D1 to D3 of PDGFRp. Preferably, the extracellular ligand binding domain of PDGFR comprises one or more mutations that increase its binding to PDGF. In a more preferred embodiment, the extracellular ligand binding domain of the PDGF receptor present in the fusion protein comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 2. In an even more preferred embodiment, the extracellular ligand binding domain of the VEGF receptor present in the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 5.

The extracellular ligand binding domain of the PDGF receptor present in the fusion protein of the present invention may be derived from any animal, such as a human or other suitable mammal such as a mouse, rabbit, rat, pig, dog or primate. In a preferred embodiment, the PDGFR is derived from a human.

As used herein, the term "Fc region" of an antibody refers to a "fragment, crystallizable" fragment of an antibody composed of two heavy chains comprising two or three constant domains, Terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region.This term includes both the native Fc region and the variant Fc region. May serve as a multimerization domain, which is a domain that facilitates the binding of the subunit to multimers, such as dimers, trimers, tetramers, etc. Preferably, The Fc region comprises one or more mutations that promote binding of the subunit to the multimer.

The Fc region of an antibody present in a fusion protein of the invention can be any homologous protein (e.g., IgG, IgM, IgA, IgD or IgE), any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgA1, IgA2, etc.), any allotype, or a knob and a hole Fc fragment. In a preferred embodiment, the Fc region of an antibody present in the fusion protein of the invention comprises the CH2 and CH3 regions of IgG1. In a more preferred embodiment, the Fc region of the antibody present in the fusion protein of the invention comprises an amino acid sequence having 90% or more homology with SEQ ID NO: 12. In a more preferred embodiment, the Fc region of the antibody present in the fusion protein of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 15.

The Fc region of an antibody present in the fusion protein of the present invention may be derived from any animal, such as a human or other suitable mammal such as a mouse, rabbit, rat, pig, dog or primate. In a preferred embodiment, the Fc region is derived from a human antibody.

According to one embodiment of the invention, the components of the fusion protein may be joined by a linking moiety such as a peptide linker. Preferably, the linker increases the flexibility of the fusion protein component, assures correct folding, minimizes steric hindrance, and does not significantly interfere with the structure of each functional component in the fusion protein. In some embodiments, the peptide linker comprises 2 to 20 amino acids. In some embodiments, the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids . Preferably, the fusion protein comprises a first linker peptide between the Fc region and the first or second peptide at the C-terminus of the fusion protein, and optionally comprises a second or first peptide at the N-terminus of the fusion protein and an Fc Lt; RTI ID = 0.0 > linker < / RTI > In a preferred embodiment of the present invention, the first linker peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 30 and 32 and the second linker peptide comprises SEQ ID NO: 18 Lt; / RTI > sequence.

According to an embodiment of the present invention, the fusion protein further comprises a signal peptide linked to the N-terminus of the fusion protein to ensure secretion of the fusion protein from the cell. Any signal peptide recognized and processed by the host cell can be used. In a preferred embodiment, the signal peptide comprises, but is not limited to, the amino acid sequence of SEQ ID NO: 34 or 36.

In a preferred embodiment, the fusion protein of the present invention has 90% or more homology with SEQ ID NO: 38, or 90% or more homology with SEQ ID NO: 40, preferably 20-766 amino acids of SEQ ID NO: 42, 21- 769 amino acid or an amino acid sequence having 90% or more homology with the 20-768 amino acid sequence of SEQ ID NO: 50. The fusion protein may comprise one or more mutations or single nucleotide polymorphisms (SNPs) of the VEGF receptor domain and / or the PDGF receptor domain. Also, one or more mutations or SNPs occurring in the Fc region of an antibody may be included according to embodiments of the present invention.

In a more preferred embodiment, the fusion protein of the invention comprises a sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 40, 20-766 amino acids of SEQ ID NO: 42, 21-769 amino acids of SEQ ID NO: 44 and 20-768 amino acids of SEQ ID NO: Selected amino acid sequence.

In a general aspect, the fusion proteins of the invention bind to VEGF and PDGF and inhibit the activity of VEGF and PDGF receptors. As used herein, the term " binds to "refers to one or more of inhibiting, blocking, neutralizing, reducing, abrogating, or interfering the activity of a ligand Of the extracellular domain of the receptor protein to the ligand. In certain embodiments, the extracellular domain of a VEGF or PDGF receptor protein binds to a ligand, i. E., VEGF or PDGF, respectively, and inhibits ligand activity by blocking the ligand from binding to other molecules such as VEGF receptors and PDGF receptors . In certain other embodiments, the extracellular domain of a VEGF or PDGF receptor protein binds to a ligand, i. E., VEGF or PDGF, respectively, to inhibit ligand activity and prevent the ligand from causing downstream signal events in the cell .

As used herein, the term "inhibit" or "inhibit" in the context of ligand activity refers to binding assays, refers to a property of an extracellular domain of a receptor protein that decreases the activity of a ligand that is analyzed by a variety of functional assays including, but not limited to, in vivo assays.

The components of a fusion protein or fusion protein disclosed herein may be selected from the group consisting of an affinity for a target binding partner (e.g., PDGF and / or VEGF family protein), a competitive binding (for example, binding of PDGF or VEGF to PDGFR or VEGFR (E.g., inhibiting activation of the PDGF or VEGF signaling pathway), inhibiting cell proliferation, inhibiting tumor growth, and inhibiting angiogenesis (e.g., inhibiting neovascularization of the choroid) But may be characterized or assessed for biological activity, including, but not limited to, In some embodiments, the components of the fusion proteins or fusion proteins disclosed herein can be assessed for biological activity in vivo or in vitro.

The invention also includes a second peptide comprising (a) a first peptide comprising an extracellular ligand binding domain of a VEGF receptor, (b) an Fc region of the antibody, and (c) an extracellular ligand binding domain of a PDGF receptor Lt; RTI ID = 0.0 > nucleic acid < / RTI > The fusion protein is arranged at the N-terminus to the C-terminus in the order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a). According to an embodiment of the present invention, the nucleic acid molecule encoding the fusion protein can be codon-optimized for expression in certain types of host cells, such as Chinese hamster ovary cells. According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the fusion protein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 37, 39, 41, 43 and 49.

According to another embodiment of the present invention, the nucleic acid molecule encoding the fusion protein may be present in an expression vector. The expression vector includes, but is not limited to, a vector for transferring a nucleic acid into a subject for expression in a specific tissue, such as a vector for expression of a recombinant protein, and a viral vector. Examples of viral vectors suitable for use with the present invention include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vectors, and the like . The vector may be a non-viral vector. Examples of non-viral vectors include, but are not limited to, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, and the like. The vector may comprise a conventional function of the expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker or an origin of replication, Lt; RTI ID = 0.0 > a < / RTI >

According to another embodiment of the present invention, the nucleic acid molecule encoding the fusion protein may be codon-optimized to improve recombinant expression from a desired host cell using methods known in the art in view of the disclosure herein .

The invention also includes a second peptide comprising (a) a first peptide comprising an extracellular ligand binding domain of a VEGF receptor, (b) an Fc region of the antibody, and (c) an extracellular ligand binding domain of a PDGF receptor Lt; RTI ID = 0.0 > a < / RTI > fusion protein; The fusion protein is arranged at the N-terminus to the C-terminus in the order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a). Host cells include, but are not limited to, host cells for recombinant protein expression and host cells for nucleic acid delivery to individuals for expression in a particular tissue. Examples of host cells suitable for use with the present invention include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK-293), and the like.

The invention also includes a second peptide comprising (a) a first peptide comprising an extracellular ligand binding domain of a VEGF receptor, (b) an Fc region of the antibody, and (c) an extracellular ligand binding domain of a PDGF receptor Lt; / RTI > fusion protein; The fusion protein is arranged at the N-terminus to the C-terminus in the order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a). In a general aspect, the method comprises: (1) culturing a host cell comprising a nucleic acid molecule encoding a fusion protein under conditions under which the fusion protein is produced; And (2) recovering the fusion protein produced by the host cell. The fusion protein can be further purified using methods known in the art.

In some embodiments, the fusion protein is expressed in a host cell and is characterized by a protein A affinity chromatography, a protein G affinity chromatography, a buffer exchange, a size exclusion chromatography Is purified therefrom using one or more of the standard purification techniques including, but not limited to, size exclusion chromatography, ultrafiltration and dialysis.

The invention also includes a second peptide comprising (a) a first peptide comprising an extracellular ligand binding domain of a VEGF receptor, (b) an Fc region of the antibody, and (c) an extracellular ligand binding domain of a PDGF receptor Lt; RTI ID = 0.0 > of: < / RTI > The fusion protein is arranged at the N-terminus to the C-terminus in the order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a). Compositions of the invention comprise a therapeutically effective amount of a fusion protein.

The term "therapeutically effective amount" means the amount of therapeutically active compound required to elicit the desired biological or clinical effect. According to an embodiment of the present invention, a "therapeutically effective amount" is an amount sufficient to achieve beneficial or desired results, including clinical results. A therapeutically effective amount can be administered in one or more administrations. In view of the disease state, an effective amount is an amount sufficient to delay the amelioration, stabilization or development of the disease. According to a specific embodiment of the present invention, a therapeutically effective amount of a fusion protein required to treat or prevent a disorder characterized by abnormal angiogenesis, such as a disease characterized by vascular permeability, edema, inflammation, retinopathy, fibrosis or cancer. It is a quantity.

In some embodiments, the pharmaceutical composition comprising the fusion protein comprises about 0.5 to about 100 mg / mL, preferably about 40 to about 80 mg / mL, such as about 40, 50, 60, 70, or 80 mg / mL, most preferably about 40 +/- 5 mg / mL, of the fusion protein. In another preferred embodiment, the fusion protein is formulated into the buffer at a protein concentration of greater than about 40 mg / mL, preferably about 80 +/- 10 mg / mL.

In certain embodiments, the buffer is a phosphate buffer having a pH of about 6.5 to 8, more preferably about 7 to 7.5, even more preferably about 7.2. The phosphate buffer may contain about 5 to 20 mM sodium phosphate, such as 5, 10, 15 or 20 mM sodium phosphate, more preferably about 10 mM sodium phosphate; About 20 to 60 mM sodium chloride, more preferably about 40 mM sodium chloride; About 1 to 10% (w / v) sucrose, more preferably about 5% w / v sucrose; And about 0.01 to 0.05% w / v of a surfactant, more preferably about 0.03% w / v polysorbate 20.

In another specific embodiment of the invention, the buffer is a histidine buffer having a pH of about 5 to 8, more preferably about 6 to 7, even more preferably about 6.8. Histidine buffer may be about 10-50 mM histidine, more preferably about 25 mM histidine, such as 10,20,30,40 or 50 mM histidine; About 10 to 30 mM sodium chloride, more preferably about 20 mM sodium chloride, such as 10, 20 or 30 mM sodium chloride; About 1 to 10% w / v male cross, more preferably about 6% w / v male cross, such as 1, 2, 4, 6, 8 or 10% w / v male cross; And about 0.01 to 0.05% w / v of a surfactant, more preferably about 0.03% w / v polysorbate 20.

The invention also includes a second peptide comprising (a) a first peptide comprising an extracellular ligand binding domain of a VEGF receptor, (b) an Fc region of the antibody, and (c) an extracellular ligand binding domain of a PDGF receptor Lt; RTI ID = 0.0 > of: < / RTI > The fusion protein is arranged at the N-terminus to the C-terminus in the order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a). Compositions of the invention comprise a therapeutically effective amount of a fusion protein.

A composition comprising a nucleic acid molecule encoding a fusion protein of the invention may comprise a delivery vehicle for introducing the nucleic acid molecule into a cell for expression of the fusion protein. Examples of nucleic acid delivery vehicles include, but are not limited to, biocompatible polymers, including liposomes, natural and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, viruses such as lipopolysaccharides, artificial viral envelopes, metal particles, and bacteria, baculoviruses, adenoviruses and retroviruses, Bacteriophages, cosmids, plasmids, fungal vectors and other recombinant vehicles typically used in the art that are described for expression in a variety of eukaryotic hosts. do.

The present invention also relates to pharmaceutical compositions of the invention for the treatment or prevention of conditions, diseases or disorders characterized by abnormal angiogenesis, such as diseases characterized by angiogenesis, vascular permeability, edema or inflammation, retinopathy, fibrosis or cancer. To the use of the composition. According to one embodiment of the present invention, a method of treating a condition, disease or disorder characterized by abnormal angiogenesis in an individual comprises administering a pharmaceutical composition of the invention to a subject in need of treatment. Any of the pharmaceutical compositions described herein may be used in the methods of the invention that comprise a pharmaceutical composition comprising a fusion protein or a pharmaceutical composition comprising a nucleic acid encoding a fusion protein. Preferably, the pharmaceutical composition of the present invention is administered via vitreous, conjunctiva, tenon, retrobulbar, or sclera in the case of ophthalmologic diseases, blood or urine in the case of systemic diseases, Lt; / RTI >

As used herein, "individual" means any animal, preferably a mammal, most preferably a human being treated or treated by a method according to an embodiment of the present invention. The term "mammal" as used herein includes any mammal. Examples of mammals include non-human primates (NHPs) such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys or apes, humans, to be.

As used herein, a "condition, disease or disorder characterized by abnormal angiogenesis" or "angiogenesis disorder" has the same meaning and includes any disorder associated with abnormal angiogenesis, including excessive, insufficient or abnormal angiogenesis . ≪ / RTI > Examples of angiogenic disorders that can be treated according to the methods of the present invention include diseases characterized by neovascularization, vascular permeability, edema or inflammation, It is not limited. These include, but are not limited to, age-related macular degeneration (e.g., mild AMD, dry AMD or lipid atrophy), proliferative and non-proliferative diabetic retinopathy, ophthalmic diseases characterized by neovascularization (e. G., Corneal neovascularization or choroidal neovascularization Ocular inflammatory and / or ocular angiogenesis, including inflammation of the central nervous system, including ophthalmopathy, diabetic retinopathy, diabetic retinopathy, rubeosis iridis and neovascular glaucoma, uveitis (e.g., uveitis or acquired uveitis) Inflammatory arthritis, osteoarthritis, autoimmune disease, or cancer. The term " autoimmune disease " includes, but is not limited to, autoimmune diseases, rheumatoid arthritis, inflammatory arthritis, In a preferred embodiment of the present invention, the angiomatous disorder that can be treated is retinopathy, fibrosis or cancer.

In another embodiment, angiogenic neurological disorders that can be treated according to the methods of the present invention include, but are not limited to, brain edema, stroke, psoriasis, asthma, generalized edema associated with burns, tumors associated with ascites and pleural effusion associated with tumors, inflammation or trauma, chronic airway inflammation, capillary leak syndrome, but are not limited to, sepsis, kidney disease associated with increased leakage of protein,

As used herein, the terms "treat," " treat "and" treatment "refer to the administration of a composition to an individual to achieve a desired therapeutic or clinical result in the individual. In one embodiment, the terms " treat ", "treating ", and" treatment "refer to administration of a pharmaceutical composition of the invention to reduce, alleviate or prevent the progression or development of angiogenic nephropathy such as vascular permeability, edema, Lt; / RTI > In another embodiment, the terms " treat, "" treat" and "treatment" refer to administering a pharmaceutical composition of the invention to inhibit or reduce corneal angiogenesis and / or leaky vasculature of the eye . In another embodiment, the terms "treat "," treating ", and "treatment" refer to the administration of a pharmaceutical composition of the invention to the progression or development of a new vessel (e.g., corneal angiogenesis) . The term "treating "," treating ", and "treating ", when used in the context of AMD, refer to preventing or reducing VEGF-induced retinal efflux and include eye scarring associated with angiogenesis, Refers to preventing or reducing fibrosis. In certain embodiments of the invention, the terms " treat, "" treating" and "treatment" when used in connection with cancer refer to reducing proliferation, undifferentiation or metastasis of cancer cells. Treatment of tumors according to the present invention includes reduction of tumor size, reduction of tumor growth and reduction of tumor metastasis. As used herein, the term "tumor" refers to abnormal tissue masses, including both benign and malignant tumors.

According to an embodiment of the present invention, the pharmaceutical composition may be administered by any method known to those skilled in the art in view of the present invention, such as topical administration, intra-vitrectomized injection, peritoneal or subconjunctival injection. In a preferred embodiment, the ophthalmic formulation is administered intravitreally. The pharmaceutical composition may be administered to any part of the eye and is preferably administered to the vitreous of the eye for the treatment of angiogenic disorders. The pharmaceutical composition may be administered to any part of the body and is preferably administered to the blood or tissue / organ for the treatment of angiogenic disorders.

Parameters such as dose, frequency and duration of administration of the pharmaceutical composition according to one embodiment of the present invention are not limited to any particular method. The optimal value of such a parameter may depend on various factors such as the subject to be treated, the particular angiogenic disease to be treated, the severity of the disease, and the like, and those skilled in the art will be able to determine the optimal value for such parameter . For example, the pharmaceutical composition may be administered once or several times a day, twice, three times, four times, or the like. Exemplary, non-limiting, dosing regimens include administration of the pharmaceutical composition once per month for a period of time in the vitreous.

In another embodiment, the invention provides a pharmaceutical composition comprising an anti-HGF receptor (HGFR), an anti-fibroblast growth factor (FGF), an anti-FGF receptor (FGFR), an anti-inflammatory agent (corticosteroid, Anti-inflammatory agents), immunomodulators, antibiotics, and anti-cancer agents.

In general terms, the present invention provides dimeric antagonists for PDGF and VEGF comprising a fusion protein of the invention. Each fusion protein in the dimer comprises any of the fusion proteins disclosed herein. In one embodiment, the dimeric fusion protein comprises two identical fusion proteins of the invention. In another embodiment, the dimeric fusion protein comprises two different fusion proteins of the invention. In another embodiment, the dimeric fusion protein is selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 40, 20-766 amino acids of SEQ ID NO: 42, 21-769 amino acids of SEQ ID NO: 44 and 20-768 amino acids of SEQ ID NO: An amino acid sequence having at least 90% homology to the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 40, 20-766 amino acids of SEQ ID NO: 42, 21-769 amino acids of SEQ ID NO: 44 and 20-768 amino acids of SEQ ID NO: / RTI > and a fusion protein having at least one fusion protein.

In another general aspect, the invention provides a protein conjugate comprising a fusion protein of the invention bound to one or more ligands selected from the group consisting of PDGF and VEGF.

Example

Example 1 is a fusion protein comprising (a) a first peptide comprising an extracellular ligand binding domain of a VEGF receptor, (b) an Fc region of an antibody, and (c) an extracellular ligand binding domain of a PDGF receptor 2 peptides; Wherein said fusion protein is arranged at the N-terminus to the C-terminus in an order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a); The fusion protein can bind to VEGF and PDGF, and can inhibit the activity of VEGF and the activity of PDGF.

Example 2 is a fusion protein according to Example 1 wherein the extracellular ligand binding domain of the VEGF receptor is capable of binding to a VEGF ligand and comprises at least one selected from the Ig-like domains D1 to D7 of the VEGF receptor can do.

Example 3 is a fusion protein according to Example 1 wherein the extracellular ligand binding domain of the PDGF receptor is capable of binding to the PDGF ligand and comprises at least one selected from the group consisting of Ig-like domains D1 to D5 of the PDGF receptor do.

Example 4 is a fusion protein according to Example 1, wherein (a) the extracellular ligand binding domain of the VEGF receptor is an Ig-like domain D2 of the first VEGFR, preferably an Ig-like domain D2 of VEGFRl, a second VEGFR Like domain D3, preferably Ig-like domain D3 of VEGFR2, (b) the Fc region of the antibody comprises the CH2 and CH3 regions of IgG1; (c) The extracellular ligand-binding domain of the PDGF receptor comprises Ig-like domains D1 to D3 of PDGFR, preferably Ig-like domains D1 to D3 of PDGFRβ.

Example 5 is a fusion protein according to Example 4, wherein (a) the extracellular ligand binding domain of the VEGF receptor comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 7; (b) the Fc region of the antibody comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 12; (c) the extracellular ligand-binding domain of the PDGF receptor comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 2.

Example 6 is a fusion protein according to Example 5, wherein (a) the extracellular ligand binding domain of a VEGF receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 7 and 10; (b) the Fc region of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 15; (c) the extracellular ligand binding domain of the PDGF receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 5.

Example 7 is a fusion protein according to any one of Examples 1 to 6 further comprising a first linker peptide between the Fc region and the first or second peptide at the C-terminus of the fusion protein, And a second linker peptide between the second or first peptide at the N-terminus and the Fc region.

Example 8 is a fusion protein according to Example 7 wherein the first linker peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 30 and 32, The linker peptide comprises the amino acid sequence of SEQ ID NO: 18.

Example 9 is a fusion protein according to any one of Examples 1 to 8, wherein the fusion protein further comprises a signal peptide linked to the N-terminus of the fusion protein.

Example 10 is a fusion protein according to Example 9, wherein the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 34 and 36.

Example 11 is a fusion protein according to any one of Examples 1 to 10 wherein the fusion protein is arranged at the N-terminus to the C-terminus in the order of (c) - (b) - (a).

Example 12 is a fusion protein comprising an amino acid sequence having 90% or more homology with SEQ ID NO: 38 or 40, or comprising 20-766 amino acids of SEQ ID NO: 42, 21-769 amino acids of SEQ ID NO: 44 or SEQ ID NO: 50 And an amino acid sequence having 90% or more homology with the 20-768 amino acid sequence.

Example 13 is a fusion protein according to Example 12 comprising the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 40, 20-766 amino acids of SEQ ID NO: 42, 21-769 amino acids of SEQ ID NO: 44 and 20-768 amino acids of SEQ ID NO: ≪ / RTI >

Example 14 is a separate nucleic acid molecule encoding a fusion protein of any of Examples 1-13.

Example 15 is the isolated nucleic acid molecule according to Example 14, wherein the fusion protein further comprises a signal peptide linked to the N-terminus of the fusion protein.

Example 16 is the isolated nucleic acid molecule according to Example 15, wherein the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 34 and 36.

Example 17 is a separate nucleic acid molecule according to any one of Examples 14 to 16, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 37, 39, 41, 43 and 49.

Example 18 is an expression vector comprising a nucleic acid molecule encoding a fusion protein of any one of Examples 1 to 13.

Example 19 is an expression vector according to Example 18, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 37, 39, 41, 43 and 49.

Example 20 is a host cell comprising a nucleic acid molecule encoding a fusion protein of any one of Examples 1-13.

Example 21 is a method for producing a fusion protein of any one of Examples 1 to 13, comprising: (1) preparing a host cell comprising a nucleic acid molecule encoding a fusion protein of any one of Examples 1 to 13, Lt; / RTI > And (2) recovering the fusion protein produced by the host cell.

Example 22 is a pharmaceutical composition comprising a fusion protein of any one of Examples 1 to 13 and a pharmaceutically acceptable carrier.

Example 23 is a pharmaceutical composition according to Example 22 wherein the composition comprises about 5-100 mM histidine, about 1-10% w / v sucrose, and about 0.005-0.1% w / v polysorbate. 0.0 > mg / ml < / RTI > of a fusion protein formulated at a pH of about 6.3 to 7.3 in a buffer containing 10-20.

Example 24 is directed to a nucleic acid molecule encoding a fusion protein of any one of Examples 1 to 13 and a lipid carrier (e.g., Lt; RTI ID = 0.0 > pharmaceutically < / RTI > acceptable carrier.

Example 25 is a pharmaceutical composition according to Example 24 wherein the composition comprises a plasmid having an expression cassette and a lipid carrier such as Lipofectamine, a compound such as polyethleneimine, ) Or an electroporation buffer. ≪ / RTI >

Example 26 is a method for treating or preventing a clinical condition selected from the group consisting of neovascularization, vascular permeability, edema and inflammation, Comprising administering to a subject in need thereof an effective amount of a fusion protein of any one of Examples 1 to 13 or a pharmaceutical composition of any one of Examples 22 to 25.

Example 27 is a method according to Example 26 wherein the fusion protein is administered as a separate protein in a pharmaceutical composition of any one of Examples 22 and 23. [

Example 28 is a method according to Example 26, wherein said fusion protein is administered in any of the pharmaceutical compositions of Examples 24 and 25.

Example 29. The method of any one of embodiments 26-28 wherein the clinical symptoms are selected from the group consisting of brain edema, stroke, cancer, psoriasis, arthritis, asthma, , Generalized edema associated with burns associated with the tumor, ascites and pleural effusion associated with tumors, inflammation or trauma, chronic airway inflammation, Related diseases such as capillary leak syndrome, sepsis, kidney disease associated with increased leakage of protein, and aged-related macular degeneration, diabetic retinopathy ), Ocular inflammation and / or ocular angiogenesis (including ocular inflammation and / or ocular angiogenesis), including uveitis and corneal neovascularization Emitter is selected.

Example 30 is a method for treating or preventing a clinical condition selected from the group consisting of choroidal neovascularization, wet age-related macular degeneration, and geographic atrophy , The method comprising administering to a subject in need thereof an effective amount of a fusion protein of any one of Examples 1 to 13 or a pharmaceutical composition of any of Examples 22 to 25.

Example 31 is a method according to Example 30 wherein the fusion protein is administered as a separate protein in the pharmaceutical composition of any one of Examples 22 and 23. [

Example 32 is a method according to Example 30, wherein the fusion protein is administered in any of the pharmaceutical compositions of Examples 24 and 25.

Example 33 is a dimeric antagonist for PDGF and VEGF comprising the fusion protein of any of Examples 1-13.

Example 34 is a protein conjugate comprising a fusion protein of any one of Examples 1 to 13 linked to one or more ligands selected from the group consisting of PDGF and VEGF.

Experimental Example

The following experimental examples of the present invention are intended to further illustrate the characteristics of the present invention. It is to be understood that the following examples do not limit the invention and that the scope of the invention is determined by the appended claims.

Experimental Example  1. Production, expression, purification and analysis of fusion proteins

PDGFR extracellular Ig-like domain (PID) (SEQ ID NO: 2) with Ig-like domain D1-D3 of PDGFRp (Heidaran et al, FASEB J. 1995; 9 (1): 140-5; Lokker et al, Like domain D2 (VEGFR-1_D2) of VEGFR-1 and Ig-like domain D3 (VEGFR-2_D3) of VEGFR-2 The VEGFR extracellular Ig-like domain (VID) (SEQ ID NO: 7) (Holash, J., et al, PNAS, 2002,99 (17): 1393-98) was incorporated into the fusion protein. In order to ensure accurate folding and minimize steric hindrance, a short variant peptide linker, GGGGGS (SEQ ID NO: 20), is inserted between the C-terminus (SEQ ID NO: 12) of the Fc region and the N- Respectively. (E. G., SEQ ID NO: 34 or SEQ ID NO: 36) to ensure secretion of the produced fusion protein 2 or fusion protein 1. The Fc region of human IgG1 is assembled to mimic the dimerization of fusion proteins, in vivo receptor dimerization, and to facilitate the easy purification of expressed fusion proteins.

The coding sequences of fusion proteins 1 and 2 having the amino acid sequences of SEQ ID NOS: 38 and 40, respectively, were transfected and expressed in human embryonic kidney cell line (HEK293F). Secreted fusion proteins were purified from cell culture supernatants using one-step Protein G chromatography. Proteins were captured with Protein G affinity column (Thermo-Fisher Scientific), eluted with low pH (3.5) buffer, and neutralized with Tris-HCl. As shown in Figures 2A-2C, greater than 90% purity was achieved using the single step purification method. Figures 2A-2C also show that the purified fusion proteins 1, 2, 3, and 5 are appropriately dimerized with predicted weights (MW ~ 180 kDa) when expressed in mammalian cells.

During development, the coding sequence of fusion protein 1, SEQ ID NO: 37, was introduced into the expression vector. The results of the competitive binding assay of purified fusion protein 1 against PDGF and the results of the PDGF-dependent BATB / 3T3 cell growth inhibition assay of fusion protein 1 indicate that there is a local effect on the binding ability of fusion protein 1, , Which has a much lower affinity for PDGF-BB (see Table 4 and Table 6).

Thus, fusion protein 2 was constructed by rearranging the directions of VID and PID. The coding sequence of fusion protein 2, i.e. SEQ ID NO: 39, was introduced into the expression vector. HEK293F host cells were transfected with expression vectors using appropriate amounts of expression vector DNA. Stable clones were selected based on protein expression levels assessed by anti-Fc ELISA and a study cell bank (RCB) was constructed from clones with optimal levels of fusion protein expression. Fusion Protein 2 RCB was used to produce shake flask protein, and the protein was purified from the culture supernatant using Protein A affinity resin. Protein was buffer exchanged with phosphate buffer with excipient. The final concentration of purified fusion protein 2 was about 20 mg / mL.

Mutagenesis of fusion protein 2 includes mutants of fusion protein 2, such as lysine residue (K528) deletion at the C-terminus of IgG1 Fc and mutation of phenylalanine to serine at the PID of fusion protein 2 To obtain fusion protein 3. The lysine was first removed using the two-step PCR mutagenesis method and the phenylalanine was changed to serine. The final construct comprises the coding sequence of SEQ ID NO: 43. CHO-S host cells were transfected with expression vectors using appropriate amounts of expression vector DNA. The original stable clones were selected based on protein expression levels assessed by anti-Fc ELISA. This was used to produce fusion protein 3 having the 21-769 amino acid sequence of SEQ ID NO: 44 (the 1 to 20 amino acids of SEQ ID NO: 44 being the signal peptide sequence cleaved during protein synthesis). Since the monoclonal antibody problem was eliminated by FACS, a round of recombination was performed. A second stable clone was selected based on the expression level and a fusion protein 4 RCB was constructed using the clone. Fusion protein 4 RCB was used for shake flask research and bioreactor production. Fusion Protein 4 RCB vials were thawed and expanded in a 1 L shake flask to produce fusion protein 4 having the same amino acid sequence as fusion protein 3. Six batches of purified proteins were prepared using protein A affinity resin (one-step purification). Protein was buffer exchanged with histidine buffer with excipient. The final concentration of fusion protein 4 was about 20 mg / mL.

To obtain a sufficient amount of fusion protein 4, a 7 L bioreactor (BR) was operated at a final working volume of about 5 L. Platform operating procedures, parameter settings, and control strategies were used, followed by badge types and supply schedules. The final product final concentration (titer) was 0.21 g / L.

The purified fusion protein sample is further subjected to clarification, protein A affinity chromatography, first ultrafiltration step, size exclusion chromatography, second ultrafiltration step, dialysis and third ultrafiltration step to obtain the final product Respectively.

In order to prepare a fusion protein 5 having the amino acid sequence 20-768 of SEQ ID NO: 50 (1 to 19 amino acids of SEQ ID NO: 50 is a signal peptide sequence cleaved during protein synthesis), the DNA sequence encoding fusion protein 5 was inserted into CHO cells Lt; / RTI > was optimized for expression in E. coli. The synthesized codon-optimized DNA having the nucleic acid sequence of SEQ ID NO: 49 was transformed into an expression vector. 72 hours before transfection, CHO Kl host cells were seeded at 2 x 10 ⁵ cells / mL in CD CHO (Gibco 12490-003) containing 4 mM glutamine (JT Baker 2078-06) and 1% HT supplement (Gibco 11067-030) . Host cells were cultured in an Infors shaker (36.5 ° C, 75% humidity, 6% CO ₂ , 110 RPM) and counted for cell density before use. Appropriate amount of expression plasmid DNA was added to the host cells (1 L or 5 L working volume) and the polymer-based transformation reagent was added. The transformed cultures were incubated for 4 hours in an Infors shaker (36.5 ° C, 75% humidity, 6% CO ₂ , 110 RPM) and proprietary feed solution was added. The transfected cultures were cultured in an Infors shaker (32 캜, 75% humidity, 6% CO ₂ , 110 RPM). Transfected cultures were harvested on day 10 after transfection. The supernatant was purified for the production of the study material. Purification procedures include purification, protein A affinity chromatography, concentration by Amicon Ultracel, size exclusion chromatography, dialysis by Slide-A-Lyzer, and final concentration by Amicon Ultracel in the formulation buffer.

Fusion protein 6 having the amino acid sequence 20-766 of SEQ ID NO: 42 was derived from fusion protein 5 by deletion of the first two amino acids (QG) of fusion protein 5, and amino acid 1 to 19 amino acids of SEQ ID NO: The truncated signal peptide sequence. The DNA insert was generated by PCR using the fusion protein 5 expression vector as a template, and the DNA sequence encoding the fusion protein 6, the nucleic acid sequence of SEQ ID NO: 41, and the silver were cloned into the expression vector. The culture and purification procedures were the same as fusion protein 5. The final concentration of fusion protein 6 was about 80 mg / mL in the designed formulation buffer (based on absorption at 280 nm).

Collectively, these data indicate that fusion proteins with activity in anti-VEGF and anti-PDGF have been generated, expressed, purified and characterized.

Experimental Example  2. Fusion protein VEGF ₁₆₅ on  Binding affinity for

In order to test the binding affinity for VEGF ₁₆₅ , a splice variant of VEGF-A of the fusion protein of the present invention, a direct binding ELISA (enzyme-linked immunosorbent assay) was performed. The synthesized VEGF traps were used as positive control 1.

The VEGF trap is a water-soluble VEGF receptor designed for treatment and is currently FDA approved for treatment of AMD. The VEGF trap contains a second Ig-like domain (D2) of VEGFRl fused to the third Ig-like domain (D3) of VEGFR2 fused to the Fc region of human IgGl (Holash, J., et al, Proc Natl Acad Sci US A. 2002 Aug 20; 99 (17): 1 1393-8). VEGF traps target VEGF-A, VEGF-B and P1GF.

To each well of a 96-well ELISA plate was added 100 [mu] l of a coating solution (1 [mu] g / ml VEGF ₁₆₅ in lx phosphate buffered saline (PBS), pH 7.2) and the plate was incubated overnight at 4 [ The wells were washed twice with 400 [mu] L of 1x PBS buffer, and excess liquid was carefully removed with a paper towel.

400 μL of blocking solution (5 g of skim milk in 100 mL 1x PBS) was added to each well, and the plate was incubated at room temperature for 1 hour. The wells were washed twice with 1x PBS buffer.

The fusion protein and the control sample were diluted 3-fold in blocking solution at the highest protein concentration of 10 mM. 100 μL of serially diluted samples was added to each well. Plates were covered and incubated at room temperature for 1 hour on a plate shaker (-100 rpm). The wells were washed three times with wash buffer (1x PBS, 0.05% Tween-20).

100 [mu] L of 1: 2500 diluted horseradish peroxidase-conjugated goat anti-human IgG Fc specific antibody in blocking solution was added to each well. The plate was sealed and incubated in a plate shaker for 1 hour at room temperature. The plates were washed three times with wash buffer.

100 μL of 3,5,3 ', 5'-tetramethylbenzidine (TMB) was added to each well, and the plate was incubated for 3 to 5 minutes to allow the reaction to take place. To stop the reaction, 100 μL of stop solution (1N HCl) was added to each well.

The optical density (OD) of each well was measured using an ELISA plate reader at an absorption wavelength of 450 nm. Absorbance was plotted against the protein concentration of the fusion protein or control and the concentration at which the signal was half the maximum effective concentration (EC ₅₀ ) was measured.

The binding affinity, as indicated by the measured EC ₅₀ value, was found to be between 0.22 and 0.93 nM for the fusion protein of the present invention. The results of the ELISA are shown in Table 1 below.

Experimental material EC ₅₀ (nM) Positive control 1 0.087 Fusion protein 1 (SEQ ID NO: 38) 0.220 Fusion protein 2 (SEQ ID NO: 40) 0.928 Fusion protein 3 (amino acid 21-769 of SEQ ID NO: 44) 0.477 Fusion protein 4 (amino acid 21-769 of SEQ ID NO: 44) 0.384 Fusion protein 5 (amino acid 20-768 of SEQ ID NO: 50) 0.388

The results of this experimental example show that the fusion proteins of the present invention, such as fusion proteins 1 to 5, bind with high affinity to VEGF ₁₆₅ . 3 is also the same.

Experimental Example  3. Fusion protein PDGF  Binding affinity for

In order to test the binding affinity of the fusion protein of the present invention for PDGF, a direct binding ELISA was performed. The synthesized PDGF trap was used as positive control 2.

The PDGF trap is a soluble PDGF receptor designed for use as a positive control. The PDGF trap contains a second Ig-like domain (D1 to D3) of PDGFRp fused to the Fc region of human IgGl (Lu et al., Am J Obstet Gynecol, 2008, 198 (4): 477.el-el0) . PDGF traps target PDGF-BB, PDGF-DD and PDGF-AB.

To each well of a 96-well ELISA plate was added 100 μL of coating solution (1 μg / mL PDGF-BB in lx phosphate buffered saline (PBS), pH 7.2) and the plate was incubated overnight at 4 ° C. The wells were washed twice with 400 [mu] L of 1x PBS buffer, and the excess liquid was carefully removed with a paper towel.

400 μL of blocking solution (1 g bovine serum albumin in 100 mL 1x PBS) was added to each well, and the plate was incubated at room temperature for 1 hour. The wells were washed twice with 1x PBS buffer.

The fusion protein and the control sample were diluted 3-fold in blocking solution at the highest protein concentration of 10 mM. 100 μL of serially diluted samples was added to each well. Plates were covered and incubated at room temperature for 1 hour on a plate shaker (-100 rpm). The wells were washed three times with wash buffer (1x PBS, 0.05% Tween-20).

100 [mu] L of 1: 2500 diluted horseradish peroxidase-conjugated goat anti-human IgG Fc specific antibody in blocking solution was added to each well. The plate was sealed and incubated in a plate shaker for 1 hour at room temperature. The plates were washed three times with wash buffer.

100 μL of 3,5,3 ', 5'-tetramethylbenzidine (TMB) was added to each well, and the plate was incubated for 3 to 5 minutes to allow the reaction to take place. To stop the reaction, 100 μL of stop solution (IN HC1) was added to each well.

The optical density (OD) of each well was measured using an ELISA plate reader at an absorption wavelength of 450 nm. Absorbance was plotted against the protein concentration of the fusion protein or control and the concentration at which the signal was half the maximum effective concentration (EC ₅₀ ) was measured.

The binding affinity, as indicated by the measured EC ₅₀ value, was found to be between 0.16 and 2.5 nM for the fusion protein of the present invention. The results of the ELISA are shown in Table 2 below.

Experimental material EC ₅₀ (nM) Positive control 1 1.354 Fusion protein 1 0.160 Fusion protein 2 0.939 Fusion protein 3 2.285 Fusion protein 4 2.538 Fusion protein 5 2.286

The results of this experimental example show that the fusion proteins of the present invention such as fusion proteins 1 to 5 bind to PDGF in high affinity. Figure 4 also shows.

Experimental Example  4. Fusion protein PDGF Competitive Combination for BB

In order to test the binding affinity of the fusion protein of the present invention for VEGF ₁₆₅ , a competitive binding assay was performed. The synthesized VEGF traps were used as positive control 1.

The fusion protein and control samples were diluted 3-fold in blocking solution with the highest protein concentration of 10 mM. Samples diluted in equal volumes were incubated overnight at room temperature with a final concentration of 5 pM VEGF-A (R & D System) with 10 pM of VEGF ₁₆₅ .

Quantikine ELISA 50 μL of a human VEGF kit (R & D Systems, Inc.) assay dilution was added to each well of a 96-well plate. 200 μL of standard, control or fusion protein was added in duplicate to the appropriate wells. The plate was sealed and incubated at room temperature for 2 hours and then washed three times with wash buffer.

200 [mu] L of the VEGF ₁₆₅ conjugate provided in the kit was added to each well, and the plate was sealed and incubated at room temperature for 2 hours. The plate was washed three times.

200 μL of the substrate solution provided in the kit was added to each well, and the plate was sealed and incubated at room temperature for 20 minutes. To stop the reaction, 50 μL of the stop solution provided in the kit was added to each well.

The OD of each well was measured using an ELISA plate reader at an absorption wavelength of 450 nm and 540 nm (or 570 nm). Unbound VEGF ₁₆₅ (free VEGF ₁₆₅ ) was plotted against the protein concentration of the fusion protein or control and the concentration (IC ₅₀ ) of the fusion protein with a 50% reduction in signal from free VEGF ₁₆₅ was measured.

The IC ₅₀ value of the fusion protein of the present invention was measured to be about 3.78 to 4.67 pM. The results of competitive binding assays are shown in Table 3 below.

Experimental material IC ₅₀ (pM) Positive control 1 5.173 Fusion protein 1 4.670 Fusion protein 2 3.780 Fusion protein 3 3.775

The results of this experimental example show that the fusion proteins of the present invention, such as fusion proteins 1, 2 and 3, bind with high affinity to VEGF. 5A and 6A.

Experimental Example  5. Fusion protein PDGF Competitive Combination for BB

In order to test the binding affinity of the fusion protein of the present invention for PDGF-BB, a competitive binding assay was performed. The synthesized PDGF trap was used as positive control 2.

The fusion protein and control samples were diluted 3-fold in blocking solution with the highest protein concentration of 10 mM. Samples diluted in equal volume were incubated at room temperature overnight with a final concentration of 10 pM with 20 pM PDGF-BB.

Quantikine ELISA Assay of human PDGF-BB kit 100 [mu] L of dilution was added to each well of a 96-well plate. 100 μL of standard, control or fusion protein was added in duplicate to the appropriate wells. The plate was sealed and incubated at room temperature for 2 hours and then washed 4 times with wash buffer.

200 [mu] L of the PDGF-BB conjugate provided in the kit was added to each well, and the plate was sealed and incubated at room temperature for 1.5 hours. The plate was washed four times.

200 μL of the substrate solution provided in the kit was added to each well, and the plate was sealed and incubated at room temperature for 20 minutes. To stop the reaction, 50 μL of the stop solution provided in the kit was added to each well.

The OD of each well was measured using an ELISA plate reader at an absorption wavelength of 450 nm and 540 nm (or 570 nm). The free PDGF-BB was plotted against the protein concentration of the fusion protein or control and the concentration (IC ₅₀ ) of the fusion protein with a 50% reduction in signal from free PDGF-BB was determined.

The IC ₅₀ value of the fusion protein of the present invention was measured to be about 0.125 to 200 nM. The results of competitive binding assays are shown in Table 4 below.

Experimental material IC ₅₀ (pM) Positive control 1 1.015 Fusion protein 1 200 Fusion protein 2 0.125 Fusion protein 3 1.371

The results of this experimental example show that the fusion proteins of the present invention, such as fusion proteins 2 and 3, bind to PDGF with high affinity. 5B and 6B.

Experimental Example  6. By fusion protein HUVEC  Proliferation inhibition

In order to test the function of the fusion protein of the present invention, human umbilical vein endothelial cell (HUVEC) proliferation assay was performed. The synthesized VEGF traps were used as positive control 1.

To each well of a 96-well ELISA plate, 100 占 퐇 of a coating solution (1% gelatin in double distilled water) was added and incubated at 37 占 폚 for 2 hours or overnight. The wells were washed twice with 1x PBS buffer.

3500 counts of human umbilical vein endothelial cells in the endothelial cell growth medium were added to each well and the plates were incubated overnight at 37 ° C.

Fusion protein samples were diluted in assay buffer (Medium-199 lx Earle's Salts, 10% bovine serum albumin, 10 mM HEPES, 1x antibiotic / antimicrobial) with the highest protein concentration of 300 nM. Fusion protein samples were mixed with VEGF ₁₆₅ (8 ng / mL) and the mixture was incubated overnight at room temperature. The wells were then washed with 200 [mu] L of 1x PBS.

VEGF was added to ₁₆₅ / sample mixture of 100 μL to each well, and the plates were incubated at 37 ℃ in 5% CO ₂ for 72 hours. After incubation, 10 μL of the MTS detection reagent (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2Htetrazolium) + Methosulfide)) was added to each well and the plate was incubated at 37 [deg.] C for 2.5 hours.

The OD of each well was measured using an ELISA plate reader at an absorption wavelength of 490 nm. Absorbance was plotted against protein concentration of the fusion protein or control and the concentration (IC ₅₀ ) at which 50% inhibition of cell proliferation was measured.

The measured IC ₅₀ was found to be between 0.058 and 0.285 nM for the fusion protein of the present invention. The results of the proliferation assay are shown in Table 5 below.

Experimental material IC ₅₀ (pM) Positive control 1 0.068 Fusion protein 1 0.058 Fusion protein 2 0.202 Fusion protein 3 0.153 Fusion protein 4 0.285 Fusion protein 5 0.112

The results of this experimental example show that the fusions of the invention, such as fusion proteins 1 to 5, inhibit VEGF-dependent growth of ITUVEC cells. 7 is also the same.

Experimental Example  7. By fusion protein BALB / 3T3 proliferation inhibition

In order to test the function of the fusion protein of the present invention, cell proliferation assay using 3T3 fibroblasts derived from BALB mouse was performed. The synthesized PDGF trap was used as positive control 2.

4000 counts of mouse 3T3 fibroblasts in DMEM (1 mM sodium pyruvate, 4 mM L-glutamine, 10% bovine serum albumin, 1x antibiotic / antimicrobial agent) were added to each well and the plates were incubated overnight at 37 ° C.

Fusion protein samples were diluted in assay buffer (1 mM sodium pyruvate, 4 mM L-glutamine, 0.5% bovine serum albumin, 1x antibiotic / antimicrobial) with the highest protein concentration of 300 nM. Fusion protein samples were mixed with PDGF (8 ng / mL) and the mixture was incubated overnight at room temperature. The wells were then washed with 200 [mu] L of 1x PBS.

The cells 37 ℃, in 5% CO ₂ for 4 hours, and the lack of assay buffer. The addition of PDGF / sample mixture of 100 μL to each well, and the plates were incubated 37 ℃, in 5% CO ₂ for 72 hours. After incubation, 10 [mu] L MTS detection reagent was added to each well and the plate was incubated at 37 [deg.] C for 2.5 hours.

The OD of each well was measured using an ELISA plate reader at an absorption wavelength of 490 nm. Absorbance was plotted against protein concentration of the fusion protein or control and the concentration (IC ₅₀ ) at which 50% inhibition of cell proliferation was measured.

The measured IC ₅₀ was found to be between 0.45 and 1000 nM for the fusion protein of the present invention. The results of the proliferation assay are shown in Table 6 below.

Experimental material IC ₅₀ (pM) Positive control 2 0.260 Fusion protein 1 1000 Fusion protein 2 0.669 Fusion protein 3 0.1992 Fusion protein 4 0.708 Fusion protein 5 0.275

The results of this experimental example show that the fusions of the invention, such as fusion proteins 1 to 5, inhibit PDGF-dependent growth of BALB / 3T3 cells. 8 is also the same.

Experimental Example  8. Netherlands by fusion protein Belted rabbit (Dutch Belted Rabbits) VEGF - Suppression of induced leakage

The effect of the fusion protein of the present invention in the prevention of vascular leakage in an in vivo model of retinal neovascularization was measured. In this model, VEGF was injected into the vitreous of rabbit eyes to induce uncontrolled angiogenesis and subsequent leakage of the retina. Avastin®, a recombinant humanized monoclonal antibody that blocks angiogenesis by inhibiting VEGF-A, and Eylea, a recombinant fusion protein composed of portions of human VEGFR1 and VEGFR2 fused to the Fc portion of human IgG1, were used as positive control groups 3 and 4 Respectively.

The Netherlands Beltled rabbits were anesthetized with isoflurane (3-5%) and treated with ophthalmic Betadine solution in the eyes. Rabbit eyes were washed with sterile physiological saline and lidocaine hydrochloride (2% injection) or proparacaine (0.5%) was added to the ocular surface.

On the first day, a fusion protein, a vehicle (negative) control, or a reference (positive) control according to an embodiment of the present invention was administered to a Dutch Belted rabbit using a BD 300 μL insulin syringe (31 gauge 5/16 inch) Dose in the vitreous. The needle was inserted through the temporal temporal lobe of the eye, about 3-4 mm in the posterior limb, and about 3-4 mm in the dorsal rectus, and 50 μL of the solution was delivered. On day 3, VEGF ₁₆₅ was injected in the same eye.

To assess leakage and torsion, Fluorescein angiograms (FAs) were performed in all dose groups after 3 days of VEGF-induction (day 6) using scales from 0 (normal) to 4 (severe).

Signs of eye irritation were demonstrated using the Draize scoring system prior to administration of the fusion protein, before VEGF induction and FA evaluation. According to the Draize analysis, all rabbit eyes were normal before the start of the study and there were no drug-related findings in the course of the study. Results scored using the Draize system were transient and may have been due to procedures related to intravenous administration as they were observed in all treatment groups.

The FAs associated with the vehicle control showed the highest mean score (2.58) for retinal vasculature leakage and torsion. The two reference positive controls had mean scores of 0.25 and 0, indicating a significant reduction in retinal vascular leakage and torsion. The fusion protein of the present invention showed an average score of 0.167 and was shown to effectively reduce VEGF-induced retinal efflux and torsion compared to the positive control. The results of in vivo assays are shown in Table 7 below.

Experimental material Capacity (㎍) score Average outflow score at 6th day Vehicle 0 12 2.583 Positive control group 3
(Avastin®) 1250 12 0.250 Positive control group 4
(Eylea®) 625 12 0 Fusion protein 4 1000 12 0.167 Fusion protein 5 1000 12 0.167

Experimental Example  9. Netherlands by fusion protein Belted Rabbit VEGF - Capacity of induced leak - Reaction suppression

The in vivo model of retinal angiogenesis was tested at various doses to determine the dose-response effect of the fusion protein of the present invention in preventing vessel leakage. In this model, VEGF ₁₆₅ was injected into the vitreous of rabbit eyes to induce uncontrolled angiogenesis and subsequent leakage of the retina.

On the first day, Dutch Beltid rabbits were injected intrathecally with varying doses of the fusion protein 5, vehicle (negative) control, or reference (positive) control according to an embodiment of the present invention. VEGF induction was performed on day 3.

To assess leakage and torsion, FA was performed in all treatment groups after 3 days of VEGF-induction (day 6) using scales from 0 (normal) to 4 (severe).

Signs of eye irritation were demonstrated using the Draize scoring system prior to administration of the fusion protein, before VEGF induction and FA evaluation. According to the Draize analysis, all rabbit eyes were normal before the start of the study and there were no drug-related findings in the course of the study. Results scored using the Draize system were transient and may have been due to procedures related to intravenous administration as they were observed in all treatment groups.

In the first VEGF-induced, FA associated with the vehicle control showed the highest mean score (3.4) with respect to retinal vasculature leakage and torsion. The two reference positive controls had a mean score of 0, signifying a significant reduction in retinal vascular leakage and torsion. The fusion protein of the present invention (fusion protein 5) showed scores of 0.08, 0.42 and 0.7, respectively, at doses of 100, 500 and 1000 μg, respectively, effectively reducing VEGF-induced retinal efflux and torsion compared to the positive control.

The results of the capacity-response in vitro analysis are shown in Table 8 below.

Experimental material Capacity (㎍) score Average outflow score at 6th day Vehicle 0 12 3.400 Positive control group 3
(Avastin®) 1250 10 0 Positive control group 4
(Eylea®) 625 12 0 Fusion protein 4 1000 12 0.167 Fusion protein 5 500 12 0.417 Fusion protein 5 100 12 0.083

Experimental Example  10. By fusion protein In rats  Reduction of laser-induced choroidal neovascularization (CNV) lesion size

The eyes of rats (Brown Norway) will be extended to 1% Cyclogyl solution and protected from light. After expansion, the rats will be anesthetized using a mixture of ketamine and xylazine. Three lesion images will be introduced into the retina of each eye using a 532 nm laser on day 1.

On day 3, the animals are anesthetized with a mixture of ketamine and xylazine, their eyes are expanded, and 5 μL of fusion protein according to an embodiment of the present invention, a vehicle (negative) control, or a reference (positive) A Hamilton syringe with a 33 gauge needle will be used to inject into both eyes of the animal.

On day 22, animals will be intraperitoneally injected with 1 [mu] L / g of 10% fluorescein sodium per body weight. To successfully identify the lesion, a Micron III Mini Animal Eye Examination (Phoenix Research) will be used to take a fundus photograph before the lesion is introduced and on day 22 after lesion burns. The lesion size will be compared and compared among the dose groups.

Experimental Example  11. By fusion protein Monkey  Laser-induced CNV  Reduction of lesion size

A laser-induced CNV model will be established in monkeys. 532 nm diode laser thermal coagulation method will be used to induce 6 ~ 9 times of burn around the macula of each eye and 0.5 mg of fusion protein of the present invention will be injected into the vitreous of the same day.

Animals will be sedated by intravenous injection of 2.5% soluble pentobitone (1 mL / kg) 20 days later. I will fix my eyelids and open my eyes and use a pigment camera to take color photographs.

Fluorescein dye (20% fluorescein sodium, 0.05 mL / kg) will be injected into the lower vein. To monitor the leakage of fluorescein associated with CNV lesions, photographs will be taken at various time points including the arterial phase, early arteriovenous fascia and some late arteriovenous foci after dye injection.

Experimental Example  12. Heterogeneity by fusion proteins Graft  Inhibition of human tumor growth in mice

Various human cancer cells such as human hepatocellular carcinoma Hep3B cells (ATCC # HB-8064) and human colorectal cancer LoVo cells (ATCC # CCL-229) can be used to establish xenograft models in nude mice.

In order to evaluate the inhibitory effect of the fusion protein of the present invention on tumor growth, tumor cells were transplanted into nude mice, and various concentrations of the fusion protein according to the examples of the present invention were administered at 0.1 to 10 mg / kg, Lt; / RTI > Tumor growth will be measured weekly for up to 7 weeks.

Experimental Example  13. Rat  And Monkey  Ocular Pharmacokinetic Evaluation of Fusion Proteins

The pharmacokinetics of the fusion proteins of the invention will be evaluated in animals. Fusion proteins ranging from 10 to 300 mg / kg will be administered to rats or monkeys via subcutaneous or intravenous injection, according to embodiments of the present invention. Blood samples will be taken at different times for up to 15 days after injection. The concentration of the fusion protein in the blood sample will be determined using an ELISA method and the pharmacokinetic parameters will be calculated.

Experimental Example  14. Rabbit and Monkey  Evaluation of Ocular Pharmacokinetics of Fusion Proteins

The pharmacokinetics of the fusion proteins of the invention will be evaluated in animals. In accordance with an embodiment of the present invention, a fusion protein ranging from 0.1 to 4 mg per eye will be administered to rabbits or monkeys via intravitreal injection. Eye tissue and blood samples will be obtained at different time points for up to 28 days after injection. Concentrations of fusion proteins in eye tissue and blood samples will be determined using ELISA methods and pharmacokinetic parameters will be calculated.

While the invention has been described in detail with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

&Lt; 110 > Allgenesis Biotherapeutics Inc.          AP BIOSCIENCES, INC. <120> Multi-targeting fusion proteins and uses thereof <130> 688947.3WO &Lt; 150 > US 62/131261 <151> 2015-03-11 <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 897 <212> DNA <213> Homo sapiens <400> 1 cagggcctgg tcgtcacacc cccggggcca gagcttgtcc tcaatgtctc cagcaccttc 60 gttctgacct gctcgggttc agctccggtg gtgtgggaac ggatgtccca ggagccccca 120 caggaaatgg ccaaggccca ggatggcacc ttctccagcg tgctcacact gaccaacctc 180 actgggctag acacgggaga atacttttgc acccacaatg actcccgtgg actggagacc 240 gatgagcgga aacggctcta catctttgtg ccagatccca ccgtgggctt cctccctaat 300 gatgccgagg aactattcat ctttctcacg gaaataactg agatcaccat tccatgccga 360 gtaacagacc cacagctggt ggtgacactg cacgagaaga aaggggacgt tgcactgcct 420 gtcccctatg atcaccaacg tggctttttt ggtatctttg aggacagaag ctacatctgc 480 aaaaccacca ttggggacag ggaggtggat tctgatgcct actatgtcta cagactccag 540 gtgtcatcca tcaacgtctc tgtgaacgca gtgcagactg tggtccgcca gggtgagaac 600 atcaccctca tgtgcattgt gatcgggaat gaggtggtca acttcgagtg gacatacccc 660 cgcaaagaaa gtgggcggct ggtggagccg gtgactgact tcctcttgga tatgccttac 720 cacatccgct ccatcctgca catccccagt gccgagttag aagactcggg gacctacacc 780 tgcaatgtga cggagagtgt gaatgaccat caggatgaaa aggccatcaa catcaccgtg 840 gttgagagcg gctacgtgcg gctcctggga gaggtgggca cactacaatt tgctgag 897 <210> 2 <211> 299 <212> PRT <213> Homo sapiens <400> 2 Gln Gly Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn Val   1 5 10 15 Ser Ser Thr Phe Val Leu Thr Cys Ser Ser Ser Ser A Pro Val Val Trp              20 25 30 Glu Arg Met Ser Glu Glu Pro Pro Gln Glu Met Ala Lys Ala Gln Asp          35 40 45 Gly Thr Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp      50 55 60 Thr Gly Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr  65 70 75 80 Asp Glu Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly                  85 90 95 Phe Leu Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile             100 105 110 Thr Glu Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val         115 120 125 Thr Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp     130 135 140 His Gln Arg Gly Phe Phe Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys 145 150 155 160 Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr Val                 165 170 175 Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val Gln             180 185 190 Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile         195 200 205 Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser     210 215 220 Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr 225 230 235 240 His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp Ser                 245 250 255 Gly Thr Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln Asp             260 265 270 Glu Lys Ala Ile Asn Ile Thr Val Val Glu Ser Gly Tyr Val Arg Leu         275 280 285 Leu Gly Glu Val Gly Thr Leu Gln Phe Ala Glu     290 295 <210> 3 <211> 891 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence for modified PID <400> 3 ctggtcgtga cacctcccgg acccgagctg gtgctcaacg tctcctccac ctttgtgctg 60 acatgcagcg gcagcgctcc tgtggtctgg gaacggatgt cccaggagcc tccccaggaa 120 atggccaagg cccaggacgg caccttttcc agcgtcctca cactcaccaa cctgacaggc 180 ctggacaccg gcgagtactt ttgcacccac aatgactcca ggggactgga aaccgatgag 240 cggaagcggc tctacatttt cgtccccgac cccaccgtcg gatttctgcc taatgacgct 300 gaagagctgt ttatcttcct gacagagatc accgaaatca ccatcccctg tcgggtcacc 360 gatccccagc tggtggtcac actgcacgag aagaagggag atgtcgccct gcctgtgcct 420 tatgaccatc agaggggctt ttccggcatt ttcgaggaca ggagctacat ctgcaaaacc 480 accatcggag accgggaggt cgacagcgat gcctattacg tctaccggct ccaggtctcc 540 tccatcaatg tgagcgtgaa tgctgtccag acagtggtcc ggcagggcga gaatatcaca 600 ctgatgtgca ttgtcattgg caacgaggtg gtcaacttcg agtggaccta tcctaggaag 660 gagagcggcc ggctcgtcga acctgtgacc gacttcctcc tggacatgcc ttaccacatt 720 cggtccatcc tgcacattcc tagcgccgag ctggaggaca gcggaaccta cacctgcaac 780 gtgaccgagt ccgtgaatga ccaccaggat gagaaggcca tcaacatcac agtcgtggag 840 agcggatacg tcaggctgct cggagaagtc ggcacactgc agttcgccga g 891 <210> 4 <211> 891 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence for modified PID <400> 4 ctggtcgtca cacccccggg gccagagctt gtcctcaatg tctccagcac cttcgttctg 60 acctgctcgg gttcagctcc ggtggtgtgg gaacggatgt cccaggagcc cccacaggaa 120 atggccaagg cccaggatgg caccttctcc agcgtgctca cactgaccaa cctcactggg 180 ctagacacgg gagaatactt ttgcacccac aatgactccc gtggactgga gaccgatgag 240 cggaaacggc tctacatctt tgtgccagat cccaccgtgg gcttcctccc taatgatgcc 300 gaggaactat tcatctttct cacggaaata actgagatca ccattccatg ccgagtaaca 360 gacccacagc tggtggtgac actgcacgag aagaaagggg acgttgcact gcctgtcccc 420 tatgatcacc aacgtggctt ttccggtatc tttgaggaca gaagctacat ctgcaaaacc 480 accattgggg acagggaggt ggattctgat gcctactatg tctacagact ccaggtgtca 540 tccatcaacg tctctgtgaa cgcagtgcag actgtggtcc gccagggtga gaacatcacc 600 ctcatgtgca ttgtgatcgg gaatgaggtg gtcaacttcg agtggacata cccccgcaaa 660 gaaagtgggc ggctggtgga gccggtgact gacttcctct tggatatgcc ttaccacatc 720 cgctccatcc tgcacatccc cagtgccgag ttagaagact cggggaccta cacctgcaat 780 gtgacggaga gtgtgaatga ccatcaggat gaaaaggcca tcaacatcac cgtggttgag 840 agcggctacg tgcggctcct gggagaggtg ggcacactac aatttgctga g 891 <210> 5 <211> 297 <212> PRT <213> Artificial Sequence <220> <223> Modified PID sequence <400> 5 Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn Val Ser Ser   1 5 10 15 Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro Val Val Trp Glu Arg              20 25 30 Met Ser Gln Glu Pro Pro Glu Glu Met Ala Lys Ala Gln Asp Gly Thr          35 40 45 Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly      50 55 60 Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu  65 70 75 80 Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu                  85 90 95 Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu             100 105 110 Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu         115 120 125 His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln     130 135 140 Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr 145 150 155 160 Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg                 165 170 175 Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val Gln Thr Val             180 185 190 Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn         195 200 205 Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg     210 215 220 Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile 225 230 235 240 Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr                 245 250 255 Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys             260 265 270 Ala Ile Asn Ile Thr Val Val Glu Ser Gly Tyr Val Arg Leu Leu Gly         275 280 285 Glu Val Gly Thr Leu Gln Phe Ala Glu     290 295 <210> 6 <211> 612 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence for VID <400> 6 gacactggta gaccttttgt tgaaatgtat tctgaaattc ctgaaattat tcatatgact 60 gaaggaagag aacttgttat tccttgtaga gttacttctc ctaatattac tgttactctt 120 aagaagtttc ctcttgatac tcttattcct gatggaaaga gaattatttg ggattctaga 180 aagggattta ttatttctaa tgctacttat aaggaaattg gacttcttac ttgtgaagct 240 actgttaatg gacatcttta taagactaat tatcttactc atagacaaac taataccatc 300 atcgacgtgg ttctgagtcc gtctcatgga attgaactat ctgttggaga aaagcttgtc 360 ttaaattgta cagcaagaac tgaactaaat gtggggattg acttcaactg ggaataccct 420 tcttcgaagc atcagcataa gaaacttgta aaccgagacc taaaaaccca gtctgggagt 480 gagatgaaga aattcttgag caccctgact atagatggtg taacccggag tgaccaagga 540 ttgtacacct gtgcagcatc cagtgggctg atgaccaaga agaacagcac atttgtcagg 600 gtccatgaaa aa 612 <210> 7 <211> 204 <212> PRT <213> Artificial Sequence <220> <223> VID sequence <400> 7 Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile   1 5 10 15 Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr              20 25 30 Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu          35 40 45 Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile      50 55 60 Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala  65 70 75 80 Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln                  85 90 95 Thr Asn Thr Ile Ile Asp Val Val Leu Ser Ser Ser His Gly Ile Glu             100 105 110 Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu         115 120 125 Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His     130 135 140 Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser 145 150 155 160 Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg                 165 170 175 Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr             180 185 190 Lys Lys Asn Ser Thr Phe Val Arg Val Val Glu Lys         195 200 <210> 8 <211> 609 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence for modified VID <400> 8 ggaaggccct tcgtggagat gtacagcgag attcctgaga ttatccacat gaccgaggga 60 cgggaactgg tgattccctg ccgggtcacc agccccaaca tcaccgtgac cctcaagaag 120 ttccccctgg acaccctgat ccctgacggc aaaaggatta tctgggacag ccggaagggc 180 tttatcatca gcaatgccac atacaaggag attggactcc tgacctgcga ggctacagtc 240 aacggacacc tgtacaagac caactacctg acccaccggc agaccaatac catcatcgac 300 gtggtgctga gccccagcca cggaattgag ctgagcgtgg gagaaaaact cgtgctcaac 360 tgcacagccc ggaccgaact caatgtcgga atcgacttca actgggaata ccccagctcc 420 aagcaccagc acaagaagct ggtcaaccgg gatctcaaga cccagtccgg cagcgaaatg 480 aagaagttcc tcagcaccct gaccatcgat ggcgtcacaa ggagcgatca gggactctac 540 acctgcgccg ctagctccgg actcatgacc aagaagaact ccacatttgt ccgggtgcac 600 gaaaagtga 609 <210> 9 <211> 606 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence for modified VID <400> 9 ggtagacctt ttgttgaaat gtattctgaa attcctgaaa ttattcatat gactgaagga 60 agagaacttg ttattccttg tagagttact tctcctaata ttactgttac tcttaagaag 120 tttcctcttg atactcttat tcctgatgga aagagaatta tttgggattc tagaaaggga 180 tttattattt ctaatgctac ttataaggaa attggacttc ttacttgtga agctactgtt 240 aatggacatc tttataagac taattatctt actcatagac aaactaatac catcatcgac 300 gtggttctga gtccgtctca tggaattgaa ctatctgttg gagaaaagct tgtcttaaat 360 tgtacagcaa gaactgaact aaatgtgggg attgacttca actgggaata cccttcttcg 420 aagcatcagc ataagaaact tgtaaaccga gacctaaaaa cccagtctgg gagtgagatg 480 aagaaattct tgagcaccct gactatagat ggtgtaaccc ggagtgacca aggattgtac 540 acctgtgcag catccagtgg gctgatgacc aagaagaaca gcacatttgt cagggtccat 600 gaaaaa 606 <210> 10 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> Modified VID sequence <400> 10 Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His   1 5 10 15 Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro              20 25 30 Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro          35 40 45 Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser      50 55 60 Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val  65 70 75 80 Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn                  85 90 95 Thr Ile Ile Asp Val Val Leu Ser Pro Ser Gly Ile Glu Leu Ser             100 105 110 Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn         115 120 125 Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His     130 135 140 Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met 145 150 155 160 Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp                 165 170 175 Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys             180 185 190 Asn Ser Thr Phe Val Arg His Val Glu Lys         195 200 <210> 11 <211> 681 <212> DNA <213> Homo sapiens <400> 11 gacaaaactc acacatgtcc accgtgtcca gcacctgaac tcctgggtgg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 12 <211> 227 <212> PRT <213> Homo sapiens <400> 12 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly   1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met              20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His          35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val      50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr  65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                  85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly Lys 225 <210> 13 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence for Fc sequence of Fusion Protein 6 <400> 13 gataagaccc acacctgtcc tccttgtcct gctcctgagc tcctcggcgg acctagcgtg 60 ttcctgtttc cccctaagcc taaagacacc ctcatgatca gccggacccc cgaggtcaca 120 tgcgtggtgg tcgacgtctc ccatgaggat cccgaggtga agttcaattg gtacgtcgac 180 ggcgtcgagg tccacaatgc caaaaccaaa ccccgggagg agcagtacaa cagcacctat 240 agggtggtca gcgtcctgac cgtgctgcac caagactggc tgaacggcaa ggagtacaaa 300 tgcaaagtca gcaataaggc cctccccgcc cccattgaga agaccatctc caaggctaag 360 ggccaaccta gggagcccca ggtgtacacc ctgcctccca gccgggatga gctgacaaag 420 aaccaggtga gcctcacctg tctggtgaag ggcttttacc cctccgatat tgccgtggag 480 tgggaaagca acggacagcc tgagaacaac tacaagacaa ccccccctgt cctggacagc 540 gatggctcct tcttcctgta cagcaagctg acagtggata agagccggtg gcagcaggga 600 aacgtcttta gctgcagcgt gatgcatgag gccctgcaca atcactacac ccagaagtcc 660 ctgtccctga gccctggc 678 <210> 14 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence for Fc sequence of Fusion Protein 4 <400> 14 gacaaaactc acacatgtcc accgtgtcca gcacctgaac tcctgggtgg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggt 678 <210> 15 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc sequence of fusion proteins <400> 15 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly   1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met              20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His          35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val      50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr  65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                  85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 16 <211> 6 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 16 gccagc 6 <210> 17 <211> 6 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 17 gctagc 6 <210> 18 <211> 2 <212> PRT <213> Artificial Sequence <220> Linker sequence <400> 18 Ala Ser   One <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 19 ggaggaggcg gtggatct 18 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> Linker sequence <400> 20 Gly Gly Gly Gly Gly Ser   1 5 <210> 21 <211> 6 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 21 ggaggc 6 <210> 22 <211> 2 <212> PRT <213> Artificial Sequence <220> Linker sequence <400> 22 Gly Gly   One <210> 23 <211> 45 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 23 ggcggcggcg gcagcggcgg aggcggatcc ggcggaggcg gctcc 45 <210> 24 <211> 15 <212> PRT <213> Artificial Sequence <220> Linker sequence <400> 24 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 25 <211> 9 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 25 ggagacacc 9 <210> 26 <211> 3 <212> PRT <213> Artificial Sequence <220> Linker sequence <400> 26 Gly Asp Thr   One <210> 27 <211> 6 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 27 ggtgga 6 <210> 28 <211> 2 <212> PRT <213> Artificial Sequence <220> Linker sequence <400> 28 Gly Gly   One <210> 29 <211> 45 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 29 ggaggcggtg gatctggtgg cggtggaagc ggaggtggag gttcc 45 <210> 30 <211> 15 <212> PRT <213> Artificial Sequence <220> Linker sequence <400> 30 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 31 <211> 9 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Coding sequence of a linker sequence <400> 31 ggagacact 9 <210> 32 <211> 3 <212> PRT <213> Artificial Sequence <220> Linker sequence <400> 32 Gly Asp Thr   One <210> 33 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of a signal peptide sequence <400> 33 atggagacag acacactcct gctatgggta ctgcttcttt gggttcccgg atccactggc 60                                                                           60 <210> 34 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide sequence <400> 34 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Gly              20 <210> 35 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of a signal peptide sequence <400> 35 atggagtttg gcctgtcctg gctcttcctc gtggctatcc tgaagggcgt gcagtgt 57 <210> 36 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide sequence <400> 36 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly   1 5 10 15 Val Gln Cys             <210> 37 <211> 2217 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of Fusion Protein 1 sequence <400> 37 gacactggaa gaccttttgt tgaaatgtat tctgaaattc ctgaaattat tcatatgact 60 gaaggaagag aacttgttat tccttgtaga gttacttctc ctaatattac tgttactctt 120 aagaagtttc ctcttgatac tcttattcct gatggaaaga gaattatttg ggattctaga 180 aagggattta ttatttctaa tgctacttat aaggaaattg gacttcttac ttgtgaagct 240 actgttaatg gacatcttta taagactaat tatcttactc atagacaaac taataccatc 300 atcgacgtgg ttctgagtcc gtctcatgga attgaactat ctgttggaga aaagcttgtc 360 ttaaattgta cagcaagaac tgaactaaat gtggggattg acttcaactg ggaataccct 420 tcttcgaagc atcagcataa gaaacttgta aaccgagacc taaaaaccca gtctgggagt 480 gagatgaaga aattcttgag caccctgact atagatggtg taacccggag tgaccaagga 540 ttgtacacct gtgcagcatc cagtgggctg atgaccaaga agaacagcac atttgtcagg 600 gtccatgaaa aagacaaaac tcacacatgt ccaccgtgtc cagcacctga actcctgggt 660 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 720 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 780 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 840 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 900 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 960 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1020 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1080 atcgccgtgg agtggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1140 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1200 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1260 acgcagaaga gcctctccct gtctccgggt aaaggtggag gaggcggtgg atccggatct 1320 cagggcctgg tcgtcacacc cccggggcca gagcttgtcc tcaatgtctc cagcaccttc 1380 gttctgacct gctcgggttc agctccggtg gtgtgggaac ggatgtccca ggagccccca 1440 caggaaatgg ccaaggccca ggatggcacc ttctccagcg tgctcacact gaccaacctc 1500 actgggctag acacgggaga atacttttgc acccacaatg actcccgtgg actggagacc 1560 gatgagcgga aacggctcta catctttgtg ccagatccca ccgtgggctt cctccctaat 1620 gatgccgagg aactattcat ctttctcacg gaaataactg agatcaccat tccatgccga 1680 gtaacagacc cacagctggt ggtgacactg cacgagaaga aaggggacgt tgcactgcct 1740 gtcccctatg atcaccaacg tggctttttt ggtatctttg aggacagaag ctacatctgc 1800 aaaaccacca ttggggacag ggaggtggat tctgatgcct actatgtcta cagactccag 1860 gtgtcatcca tcaacgtctc tgtgaacgca gtgcagactg tggtccgcca gggtgagaac 1920 atcaccctca tgtgcattgt gatcgggaat gaggtggtca acttcgagtg gacatacccc 1980 cgcaaagaaa gtgggcggct ggtggagccg gtgactgact tcctcttgga tatgccttac 2040 cacatccgct ccatcctgca catccccagt gccgagttag aagactcggg gacctacacc 2100 tgcaatgtga cggagagtgt gaatgaccat caggatgaaa aggccatcaa catcaccgtg 2160 gttgagagcg gctacgtgcg gctcctggga gaggtgggca cactacaatt tgctgag 2217 <210> 38 <211> 739 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein 1 sequence <400> 38 Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile   1 5 10 15 Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr              20 25 30 Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu          35 40 45 Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile      50 55 60 Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala  65 70 75 80 Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln                  85 90 95 Thr Asn Thr Ile Ile Asp Val Val Leu Ser Ser Ser His Gly Ile Glu             100 105 110 Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu         115 120 125 Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His     130 135 140 Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser 145 150 155 160 Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg                 165 170 175 Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr             180 185 190 Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr His         195 200 205 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val     210 215 220 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 225 230 235 240 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu                 245 250 255 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys             260 265 270 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser         275 280 285 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys     290 295 300 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 305 310 315 320 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro                 325 330 335 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu             340 345 350 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn         355 360 365 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser     370 375 380 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 385 390 395 400 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu                 405 410 415 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly             420 425 430 Gly Gly Gly Gly Gly Ser Gly Ser Gln Gly Leu Val Val Thr Pro Pro         435 440 445 Gly Pro Glu Leu Val Leu Asn Val Ser Ser Thr Phe Val Leu Thr Cys     450 455 460 Ser Gly Ser Ala Pro Val Val Trp Glu Arg Met Ser Gln Glu Pro Pro 465 470 475 480 Gln Glu Met Ala Lys Ala Gln Asp Gly Thr Phe Ser Ser Val Leu Thr                 485 490 495 Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly Glu Tyr Phe Cys Thr His             500 505 510 Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr Ile         515 520 525 Phe Val Pro Asp Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu Glu     530 535 540 Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys Arg 545 550 555 560 Val Thr Asp Pro Gln Leu Val Val Thr Leu His Glu Lys Lys Gly Asp                 565 570 575 Val Ala Leu Pro Val Pro Tyr Asp His Gln Arg Gly Phe Phe Gly Ile             580 585 590 Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg Glu         595 600 605 Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser Ile     610 615 620 Asn Val Ser Val Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu Asn 625 630 635 640 Ile Thr Leu Met Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe Glu                 645 650 655 Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val Thr             660 665 670 Asp Phe Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile Leu His Ile         675 680 685 Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val Thr     690 695 700 Glu Ser Val Asn Asp His Gln Asp Glu Lys Ala Ile Asn Ile Thr Val 705 710 715 720 Val Glu Ser Gly Tyr Val Arg Leu Leu Gly Glu Val Gly Thr Leu Gln                 725 730 735 Phe Ala Glu             <210> 39 <211> 2250 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of Fusion Protein 2 sequence <400> 39 cagggcctgg tcgtcacacc cccggggcca gagcttgtcc tcaatgtctc cagcaccttc 60 gttctgacct gctcgggttc agctccggtg gtgtgggaac ggatgtccca ggagccccca 120 caggaaatgg ccaaggccca ggatggcacc ttctccagcg tgctcacact gaccaacctc 180 actgggctag acacgggaga atacttttgc acccacaatg actcccgtgg actggagacc 240 gatgagcgga aacggctcta catctttgtg ccagatccca ccgtgggctt cctccctaat 300 gatgccgagg aactattcat ctttctcacg gaaataactg agatcaccat tccatgccga 360 gtaacagacc cacagctggt ggtgacactg cacgagaaga aaggggacgt tgcactgcct 420 gtcccctatg atcaccaacg tggctttttt ggtatctttg aggacagaag ctacatctgc 480 aaaaccacca ttggggacag ggaggtggat tctgatgcct actatgtcta cagactccag 540 gtgtcatcca tcaacgtctc tgtgaacgca gtgcagactg tggtccgcca gggtgagaac 600 atcaccctca tgtgcattgt gatcgggaat gaggtggtca acttcgagtg gacatacccc 660 cgcaaagaaa gtgggcggct ggtggagccg gtgactgact tcctcttgga tatgccttac 720 cacatccgct ccatcctgca catccccagt gccgagttag aagactcggg gacctacacc 780 tgcaatgtga cggagagtgt gaatgaccat caggatgaaa aggccatcaa catcaccgtg 840 gttgagagcg gctacgtgcg gctcctggga gaggtgggca cactacaatt tgctgaggct 900 agcgacaaaa ctcacacatg tccaccgtgt ccagcacctg aactcctggg tggaccgtca 960 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 1020 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 1080 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 1140 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1200 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1260 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1320 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1380 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1440 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1500 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1560 agcctctccc tgtctccggg taaaggtgga ggaggcggtg gatctggtgg cggtggaagc 1620 ggaggtggag gttccggaga cactggtaga ccttttgttg aaatgtattc tgaaattcct 1680 gaaattattc atatgactga aggaagagaa cttgttattc cttgtagagt tacttctcct 1740 aatattactg ttactcttaa gaagtttcct cttgatactc ttattcctga tggaaagaga 1800 attatttggg attctagaaa gggatttatt atttctaatg ctacttataa ggaaattgga 1860 cttcttactt gtgaagctac tgttaatgga catctttata agactaatta tcttactcat 1920 agacaaacta ataccatcat cgacgtggtt ctgagtccgt ctcatggaat tgaactatct 1980 gttggagaaa agcttgtctt aaattgtaca gcaagaactg aactaaatgt ggggattgac 2040 ttcaactggg aatacccttc ttcgaagcat cagcataaga aacttgtaaa ccgagaccta 2100 aaaacccagt ctgggagtga gatgaagaaa ttcttgagca ccctgactat agatggtgta 2160 acccggagtg accaaggatt gtacacctgt gcagcatcca gtgggctgat gaccaagaag 2220 aacagcacat ttgtcagggt ccatgaaaaa 2250 <210> 40 <211> 750 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein 2 sequence <400> 40 Gln Gly Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn Val   1 5 10 15 Ser Ser Thr Phe Val Leu Thr Cys Ser Ser Ser Ser A Pro Val Val Trp              20 25 30 Glu Arg Met Ser Glu Glu Pro Pro Gln Glu Met Ala Lys Ala Gln Asp          35 40 45 Gly Thr Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp      50 55 60 Thr Gly Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr  65 70 75 80 Asp Glu Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly                  85 90 95 Phe Leu Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile             100 105 110 Thr Glu Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val         115 120 125 Thr Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp     130 135 140 His Gln Arg Gly Phe Phe Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys 145 150 155 160 Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr Val                 165 170 175 Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val Gln             180 185 190 Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile         195 200 205 Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser     210 215 220 Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr 225 230 235 240 His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp Ser                 245 250 255 Gly Thr Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln Asp             260 265 270 Glu Lys Ala Ile Asn Ile Thr Val Val Glu Ser Gly Tyr Val Arg Leu         275 280 285 Leu Gly Glu Val Gly Thr Leu Gln Phe Ala Glu Ala Ser Asp Lys Thr     290 295 300 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 305 310 315 320 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 325 330 335 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp Pro             340 345 350 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         355 360 365 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val     370 375 380 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 385 390 395 400 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr                 405 410 415 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             420 425 430 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys         435 440 445 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     450 455 460 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 465 470 475 480 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser                 485 490 495 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             500 505 510 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         515 520 525 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly     530 535 540 Ser Gly Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro 545 550 555 560 Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg                 565 570 575 Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp             580 585 590 Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly         595 600 605 Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys     610 615 620 Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His 625 630 635 640 Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly                 645 650 655 Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg             660 665 670 Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser         675 680 685 Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser     690 695 700 Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val 705 710 715 720 Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu                 725 730 735 Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys             740 745 750 <210> 41 <211> 2301 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of Fusion Protein 6 sequence <400> 41 atggagtttg gcctgtcctg gctcttcctc gtggctatcc tgaagggcgt gcagtgtctg 60 gtcgtgacac ctcccggacc cgagctggtg ctcaacgtct cctccacctt tgtgctgaca 120 tgcagcggca gcgctcctgt ggtctgggaa cggatgtccc aggagcctcc ccaggaaatg 180 gccaaggccc aggacggcac cttttccagc gtcctcacac tcaccaacct gacaggcctg 240 gacaccggcg agtacttttg cacccacaat gactccaggg gactggaaac cgatgagcgg 300 aagcggctct acattttcgt ccccgacccc accgtcggat ttctgcctaa tgacgctgaa 360 gagctgttta tcttcctgac agagatcacc gaaatcacca tcccctgtcg ggtcaccgat 420 ccccagctgg tggtcacact gcacgagaag aagggagatg tcgccctgcc tgtgccttat 480 gaccatcaga ggggcttttc cggcattttc gaggacagga gctacatctg caaaaccacc 540 atcggagacc gggaggtcga cagcgatgcc tattacgtct accggctcca ggtctcctcc 600 atcaatgtga gcgtgaatgc tgtccagaca gtggtccggc agggcgagaa tatcacactg 660 atgtgcattg tcattggcaa cgaggtggtc aacttcgagt ggacctatcc taggaaggag 720 agcggccggc tcgtcgaacc tgtgaccgac ttcctcctgg acatgcctta ccacattcgg 780 tccatcctgc acattcctag cgccgagctg gaggacagcg gaacctacac ctgcaacgtg 840 accgagtccg tgaatgacca ccaggatgag aaggccatca acatcacagt cgtggagagc 900 ggatacgtca ggctgctcgg agaagtcggc acactgcagt tcgccgaggc cagcgataag 960 acccacacct gtcctccttg tcctgctcct gagctcctcg gcggacctag cgtgttcctg 1020 tttcccccta agcctaaaga caccctcatg atcagccgga cccccgaggt cacatgcgtg 1080 gtggtcgacg tctcccatga ggatcccgag gtgaagttca attggtacgt cgacggcgtc 1140 gaggtccaca atgccaaaac caaaccccgg gaggagcagt acaacagcac ctatagggtg 1200 gtcagcgtcc tgaccgtgct gcaccaagac tggctgaacg gcaaggagta caaatgcaaa 1260 gtcagcaata aggccctccc cgcccccatt gagaagacca tctccaaggc taagggccaa 1320 cctagggagc cccaggtgta caccctgcct cccagccggg atgagctgac aaagaaccag 1380 gtgagcctca cctgtctggt gaagggcttt tacccctccg atattgccgt ggagtgggaa 1440 agcaacggac agcctgagaa caactacaag acaacccccc ctgtcctgga cagcgatggc 1500 tccttcttcc tgtacagcaa gctgacagtg gataagagcc ggtggcagca gggaaacgtc 1560 tttagctgca gcgtgatgca tgaggccctg cacaatcact acacccagaa gtccctgtcc 1620 ctgagccctg gcggaggcgg cggcggcggc agcggcggag gcggatccgg cggaggcggc 1680 tccggagaca ccggaaggcc cttcgtggag atgtacagcg agattcctga gattatccac 1740 atgaccgagg gacgggaact ggtgattccc tgccgggtca ccagccccaa catcaccgtg 1800 accctcaaga agttccccct ggacaccctg atccctgacg gcaaaaggat tatctgggac 1860 agccggaagg gctttatcat cagcaatgcc acatacaagg agattggact cctgacctgc 1920 gaggctacaga tcaacggaca cctgtacaag accaactacc tgacccaccg gcagaccaat 1980 accatcatcg acgtggtgct gagccccagc cacggaattg agctgagcgt gggagaaaaa 2040 ctcgtgctca actgcacagc ccggaccgaa ctcaatgtcg gaatcgactt caactgggaa 2100 taccccagct ccaagcacca gcacaagaag ctggtcaacc gggatctcaa gacccagtcc 2160 ggcagcgaaa tgaagaagtt cctcagcacc ctgaccatcg atggcgtcac aaggagcgat 2220 cagggactct acacctgcgc cgctagctcc ggactcatga ccaagaagaa ctccacattt 2280 gtccgggtgc acgaaaagtg a 2301 <210> 42 <211> 766 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein 6 sequence <400> 42 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly   1 5 10 15 Val Gln Cys Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn              20 25 30 Val Ser Ser Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro Val Val          35 40 45 Trp Glu Arg Met Ser Glu Glu Pro Pro Gln Glu Met Ala Lys Ala Gln      50 55 60 Asp Gly Thr Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu  65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu                  85 90 95 Thr Asp Glu Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val             100 105 110 Gly Phe Leu Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu         115 120 125 Ile Thr Glu Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val     130 135 140 Val Thr Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr 145 150 155 160 Asp His Gln Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile                 165 170 175 Cys Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr             180 185 190 Val Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val         195 200 205 Gln Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val     210 215 220 Ile Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu 225 230 235 240 Ser Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro                 245 250 255 Tyr His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp             260 265 270 Ser Gly Thr Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln         275 280 285 Asp Glu Lys Ala Ile Asn Ile Thr Val Val Glu Ser Gly Tyr Val Arg     290 295 300 Leu Leu Gly Glu Val Gly Thr Leu Gln Phe Ala Glu Ala Ser Asp Lys 305 310 315 320 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro                 325 330 335 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             340 345 350 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp         355 360 365 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     370 375 380 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 385 390 395 400 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu                 405 410 415 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys             420 425 430 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         435 440 445 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr     450 455 460 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 465 470 475 480 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu                 485 490 495 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             500 505 510 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         515 520 525 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     530 535 540 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 545 550 555 560 Ser Gly Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro                 565 570 575 Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg             580 585 590 Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp         595 600 605 Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly     610 615 620 Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys 625 630 635 640 Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His                 645 650 655 Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly             660 665 670 Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg         675 680 685 Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser     690 695 700 Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser 705 710 715 720 Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val                 725 730 735 Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu             740 745 750 Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys         755 760 765 <210> 43 <211> 2307 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of Fusion Proteins 3 and 4 sequence <400> 43 atggagacag acacactcct gctatgggta ctgcttcttt gggttcccgg atccactggc 60 cagggcctgg tcgtcacacc cccggggcca gagcttgtcc tcaatgtctc cagcaccttc 120 gttctgacct gctcgggttc agctccggtg gtgtgggaac ggatgtccca ggagccccca 180 caggaaatgg ccaaggccca ggatggcacc ttctccagcg tgctcacact gaccaacctc 240 actgggctag acacgggaga atacttttgc acccacaatg actcccgtgg actggagacc 300 cctccctaat 360 gatgccgagg aactattcat ctttctcacg gaaataactg agatcaccat tccatgccga 420 gtaacagacc cacagctggt ggtgacactg cacgagaaga aaggggacgt tgcactgcct 480 gtcccctatg atcaccaacg tggcttttcc ggtatctttg aggacagaag ctacatctgc 540 aaaaccacca ttggggacag ggaggtggat tctgatgcct actatgtcta cagactccag 600 gtgtcatcca tcaacgtctc tgtgaacgca gtgcagactg tggtccgcca gggtgagaac 660 atcaccctca tgtgcattgt gatcgggaat gaggtggtca acttcgagtg gacatacccc 720 cgcaaagaaa gtgggcggct ggtggagccg gtgactgact tcctcttgga tatgccttac 780 cacatccgct ccatcctgca catccccagt gccgagttag aagactcggg gacctacacc 840 tgcaatgtga cggagagtgt gaatgaccat caggatgaaa aggccatcaa catcaccgtg 900 gttgagagcg gctacgtgcg gctcctggga gaggtgggca cactacaatt tgctgaggct 960 agcgacaaaa ctcacacatg tccaccgtgt ccagcacctg aactcctggg tggaccgtca 1020 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 1080 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 1140 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 1200 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1260 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1320 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1380 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1440 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1500 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1560 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1620 agcctctccc tgtctccggg tggtggagga ggcggtggat ctggtggcgg tggaagcgga 1680 ggtggaggtt ccggagacac tggtagacct tttgttgaaa tgtattctga aattcctgaa 1740 attattcata tgactgaagg aagagaactt gttattcctt gtagagttac ttctcctaat 1800 attactgtta ctcttaagaa gtttcctctt gatactctta ttcctgatgg aaagagaatt 1860 atttgggatt ctagaaaggg atttattatt tctaatgcta cttataagga aattggactt 1920 cttacttgtg aagctactgt taatggacat ctttataaga ctaattatct tactcataga 1980 caaactaata ccatcatcga cgtggttctg agtccgtctc atggaattga actatctgtt 2040 ggagaaaagc ttgtcttaaa ttgtacagca agaactgaac taaatgtggg gattgacttc 2100 aactgggaat acccttcttc gaagcatcag cataagaaac ttgtaaaccg agacctaaaa 2160 acccagtctg ggagtgagat gaagaaattc ttgagcaccc tgactataga tggtgtaacc 2220 cggagtgacc aaggattgta cacctgtgca gcatccagtg ggctgatgac caagaagaac 2280 agcacatttg tcagggtcca tgaaaaa 2307 <210> 44 <211> 769 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein 3 or 4 plus a signal peptide sequence <400> 44 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Gly Gln Gly Leu Val Val Thr Pro Pro Gly Pro Glu Leu              20 25 30 Val Leu Asn Val Ser Ser Thr Phe Val Leu Thr Cys Ser Gly Ser Ala          35 40 45 Pro Val Val Trp Glu Arg Met Ser Glu Glu Pro Pro Gln Glu Met Ala      50 55 60 Lys Ala Gln Asp Gly Thr Phe Ser Ser Val Leu Thr Leu Thr Asn Leu  65 70 75 80 Thr Gly Leu Asp Thr Gly Glu Tyr Phe Cys Thr His Asn Asp Ser Arg                  85 90 95 Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp             100 105 110 Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe         115 120 125 Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys Arg Val Thr Asp Pro     130 135 140 Gln Leu Val Val Thr Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro 145 150 155 160 Val Pro Tyr Asp His Gln Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg                 165 170 175 Ser Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp             180 185 190 Ala Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val         195 200 205 Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met     210 215 220 Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro 225 230 235 240 Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu                 245 250 255 Asp Met Pro Tyr His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu             260 265 270 Leu Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val Thr Glu Ser Val Asn         275 280 285 Asp His Gln Asp Glu Lys Ala Ile Asn Ile Thr Val Val Glu Ser Gly     290 295 300 Tyr Val Arg Leu Leu Gly Glu Val Gly Thr Leu Gln Phe Ala Glu Ala 305 310 315 320 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                 325 330 335 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu             340 345 350 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser         355 360 365 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu     370 375 380 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 385 390 395 400 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn                 405 410 415 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro             420 425 430 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln         435 440 445 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val     450 455 460 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 465 470 475 480 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro                 485 490 495 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr             500 505 510 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val         515 520 525 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu     530 535 540 Ser Pro Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 545 550 555 560 Gly Gly Gly Ser Gly Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser                 565 570 575 Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile             580 585 590 Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe         595 600 605 Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser     610 615 620 Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu 625 630 635 640 Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr                 645 650 655 Leu Thr His Arg Gln Thr Asn Thr Ile Asp Val Val Leu Ser Pro             660 665 670 Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys         675 680 685 Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr     690 695 700 Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys 705 710 715 720 Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile                 725 730 735 Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser             740 745 750 Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu         755 760 765 Lys     <210> 45 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of VEGF Trap sequence <400> 45 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg gtcgactggc 60 gacactggaa gaccttttgt tgaaatgtat tctgaaattc ctgaaattat tcatatgact 120 gaaggaagag aacttgttat tccttgtaga gttacttctc ctaatattac tgttactctt 180 aagaagtttc ctcttgatac tcttattcct gatggaaaga gaattatttg ggattctaga 240 aagggattta ttatttctaa tgctacttat aaggaaattg gacttcttac ttgtgaagct 300 actgttaatg gacatcttta taagactaat tatcttactc atagacaaac taataccatc 360 atcgacgtgg ttctgagtcc gtctcatgga attgaactat ctgttggaga aaagcttgtc 420 ttaaattgta cagcaagaac tgaactaaat gtggggattg acttcaactg ggaataccct 480 tcttcgaagc atcagcataa gaaacttgta aaccgagacc taaaaaccca gtctgggagt 540 gagatgaaga aattcttgag caccctgact atagatggtg taacccggag tgaccaagga 600 ttgtacacct gtgcagcatc cagtgggctg atgaccaaga agaacagcac atttgtcagg 660 gtccatgaaa aagacaaaac tcacacatgt ccaccgtgtc cagcacctga actcctgggt 720 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1080 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320 acgcagaaga gcctctccct gtctccgggt aaa 1353 <210> 46 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> VEGF Trap sequence Plus a signal sequence <400> 46 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Gly Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu              20 25 30 Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro          35 40 45 Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro      50 55 60 Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg  65 70 75 80 Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu                  85 90 95 Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu             100 105 110 Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser         115 120 125 His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr     130 135 140 Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro 145 150 155 160 Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr                 165 170 175 Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp             180 185 190 Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser         195 200 205 Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys     210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser         355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435 440 445 Pro Gly Lys     450 <210> 47 <211> 1644 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of PDGF Trap sequence <400> 47 atggagacag acacactcct gctatgggta ctgcttcttt gggttcccgg atccactggc 60 cagggcctgg tcgtcacacc cccggggcca gagcttgtcc tcaatgtctc cagcaccttc 120 gttctgacct gctcgggttc agctccggtg gtgtgggaac ggatgtccca ggagccccca 180 caggaaatgg ccaaggccca ggatggcacc ttctccagcg tgctcacact gaccaacctc 240 actgggctag acacgggaga atacttttgc acccacaatg actcccgtgg actggagacc 300 cctccctaat 360 gatgccgagg aactattcat ctttctcacg gaaataactg agatcaccat tccatgccga 420 gtaacagacc cacagctggt ggtgacactg cacgagaaga aaggggacgt tgcactgcct 480 gtcccctatg atcaccaacg tggctttttt ggtatctttg aggacagaag ctacatctgc 540 aaaaccacca ttggggacag ggaggtggat tctgatgcct actatgtcta cagactccag 600 gtgtcatcca tcaacgtctc tgtgaacgca gtgcagactg tggtccgcca gggtgagaac 660 atcaccctca tgtgcattgt gatcgggaat gaggtggtca acttcgagtg gacatacccc 720 cgcaaagaaa gtgggcggct ggtggagccg gtgactgact tcctcttgga tatgccttac 780 cacatccgct ccatcctgca catccccagt gccgagttag aagactcggg gacctacacc 840 tgcaatgtga cggagagtgt gaatgaccat caggatgaaa aggccatcaa catcaccgtg 900 gttgagagcg gctacgtgcg gctcctggga gaggtgggca cactacaatt tgctgaggct 960 agcgacaaaa ctcacacatg tccaccgtgt ccagcacctg aactcctggg tggaccgtca 1020 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 1080 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 1140 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 1200 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1260 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1320 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1380 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1440 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1500 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1560 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1620 agcctctccc tgtctccggg taaa 1644 <210> 48 <211> 548 <212> PRT <213> Artificial Sequence <220> <223> PDGF Trap sequence plus a signal sequence <400> 48 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Gly Gln Gly Leu Val Val Thr Pro Pro Gly Pro Glu Leu              20 25 30 Val Leu Asn Val Ser Ser Thr Phe Val Leu Thr Cys Ser Gly Ser Ala          35 40 45 Pro Val Val Trp Glu Arg Met Ser Glu Glu Pro Pro Gln Glu Met Ala      50 55 60 Lys Ala Gln Asp Gly Thr Phe Ser Ser Val Leu Thr Leu Thr Asn Leu  65 70 75 80 Thr Gly Leu Asp Thr Gly Glu Tyr Phe Cys Thr His Asn Asp Ser Arg                  85 90 95 Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp             100 105 110 Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe         115 120 125 Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys Arg Val Thr Asp Pro     130 135 140 Gln Leu Val Val Thr Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro 145 150 155 160 Val Pro Tyr Asp His Gln Arg Gly Phe Phe Gly Ile Phe Glu Asp Arg                 165 170 175 Ser Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp             180 185 190 Ala Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val         195 200 205 Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met     210 215 220 Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro 225 230 235 240 Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu                 245 250 255 Asp Met Pro Tyr His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu             260 265 270 Leu Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val Thr Glu Ser Val Asn         275 280 285 Asp His Gln Asp Glu Lys Ala Ile Asn Ile Thr Val Val Glu Ser Gly     290 295 300 Tyr Val Arg Leu Leu Gly Glu Val Gly Thr Leu Gln Phe Ala Glu Ala 305 310 315 320 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                 325 330 335 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu             340 345 350 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser         355 360 365 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu     370 375 380 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 385 390 395 400 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn                 405 410 415 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro             420 425 430 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln         435 440 445 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val     450 455 460 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 465 470 475 480 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro                 485 490 495 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr             500 505 510 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val         515 520 525 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu     530 535 540 Ser Pro Gly Lys 545 <210> 49 <211> 2250 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of Fusion Protein 5 sequence <400> 49 cagggcctgg tcgtgacacc tcccggaccc gagctggtgc tcaacgtctc ctccaccttt 60 gtgctgacat gcagcggcag cgctcctgtg gtctgggaac ggatgtccca ggagcctccc 120 caggaaatgg ccaaggccca ggacggcacc ttttccagcg tcctcacact caccaacctg 180 acaggcctgg acaccggcga gtacttttgc acccacaatg actccagggg actggaaacc 240 cctgcctgat gacgctgaag agctgtttat cttcctgaca gagatcaccg aaatcaccat cccctgtcgg 360 gtcaccgatc cccagctggt ggtcacactg cacgagaaga agggagatgt cgccctgcct 420 gtgccttatg accatcagag gggcttttcc ggcattttcg aggacaggag ctacatctgc 480 aaaaccacca tcggagaccg ggaggtcgac agcgatgcct attacgtcta ccggctccag 540 gtctcctcca tcaatgtgag cgtgaatgct gtccagacag tggtccggca gggcgagaat 600 atcacactga tgtgcattgt cattggcaac gaggtggtca acttcgagtg gacctatcct 660 aggaaggaga gcggccggct cgtcgaacct gtgaccgact tcctcctgga catgccttac 720 cacattcggt ccatcctgca cattcctagc gccgagctgg aggacagcgg aacctacacc 780 tgcaacgtga ccgagtccgt gaatgaccac caggatgaga aggccatcaa catcacagtc 840 gtggagagcg gatacgtcag gctgctcgga gaagtcggca cactgcagtt cgccgaggcc 900 agcgataaga cccacacctg tcctccttgt cctgctcctg agctcctcgg cggacctagc 960 gtgttcctgt ttccccctaa gcctaaagac accctcatga tcagccggac ccccgaggtc 1020 acatgcgtgg tggtcgacgt ctcccatgag gatcccgagg tgaagttcaa ttggtacgtc 1080 gacggcgtcg aggtccacaa tgccaaaacc aaaccccggg aggagcagta caacagcacc 1140 tatagggtgg tcagcgtcct gaccgtgctg caccaagact ggctgaacgg caaggagtac 1200 aaatgcaaag tcagcaataa ggccctcccc gcccccattg agaagaccat ctccaaggct 1260 aagggccaac ctagggagcc ccaggtgtac accctgcctc ccagccggga tgagctgaca 1320 aagaaccagg tgagcctcac ctgtctggtg aagggctttt acccctccga tattgccgtg 1380 gagtgggaaa gcaacggaca gcctgagaac aactacaaga caaccccccc tgtcctggac 1440 agcgatggct ccttcttcct gtacagcaag ctgacagtgg ataagagccg gtggcagcag 1500 ggaaacgtct ttagctgcag cgtgatgcat gaggccctgc acaatcacta cacccagaag 1560 tccctgtccc tgagccctgg cggaggcggc ggcggcggca gcggcggagg cggatccggc 1620 ggaggcggct ccggagacac cggaaggccc ttcgtggaga tgtacagcga gattcctgag 1680 attatccaca tgaccgaggg acgggaactg gtgattccct gccgggtcac cagccccaac 1740 atcaccgtga ccctcaagaa gttccccctg gacaccctga tccctgacgg caaaaggatt 1800 atctgggaca gccggaaggg ctttatcatc agcaatgcca catacaagga gattggactc 1860 ctgacctgcg aggctacagt caacggacac ctgtacaaga ccaactacct gacccaccgg 1920 cagaccaata ccatcatcga cgtggtgctg agccccagcc acggaattga gctgagcgtg 1980 ggagaaaaac tcgtgctcaa ctgcacagcc cggaccgaac tcaatgtcgg aatcgacttc 2040 aactgggaat accccagctc caagcaccag cacaagaagc tggtcaaccg ggatctcaag 2100 acccagtccg gcagcgaaat gaagaagttc ctcagcaccc tgaccatcga tggcgtcaca 2160 aggagcgatc agggactcta cacctgcgcc gctagctccg gactcatgac caagaagaac 2220 tccacatttg tccgggtgca cgaaaagtga 2250 <210> 50 <211> 768 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein 5 plus a signal peptide sequence <400> 50 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly   1 5 10 15 Val Gln Cys Gln Gly Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val              20 25 30 Leu Asn Val Ser Ser Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro          35 40 45 Val Val Trp Glu Arg Met Ser Glu Glu Pro Pro Gln Glu Met Ala Lys      50 55 60 Ala Gln Asp Gly Thr Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr  65 70 75 80 Gly Leu Asp Thr Gly Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly                  85 90 95 Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro             100 105 110 Thr Val Gly Phe Leu Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu         115 120 125 Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln     130 135 140 Leu Val Val Thr Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro Val 145 150 155 160 Pro Tyr Asp His Gln Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser                 165 170 175 Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala             180 185 190 Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn         195 200 205 Ala Val Gln Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys     210 215 220 Ile Val Ile Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg 225 230 235 240 Lys Glu Ser Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp                 245 250 255 Met Pro Tyr His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu             260 265 270 Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp         275 280 285 His Gln Asp Glu Lys Ala Ile Asn Ile Thr Val Val Glu Ser Gly Tyr     290 295 300 Val Arg Leu Leu Gly Glu Val Gly Thr Leu Gln Phe Ala Glu Ala Ser 305 310 315 320 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly                 325 330 335 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             340 345 350 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         355 360 365 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     370 375 380 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 385 390 395 400 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 405 410 415 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             420 425 430 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         435 440 445 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     450 455 460 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 465 470 475 480 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 485 490 495 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             500 505 510 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         515 520 525 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     530 535 540 Pro Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 545 550 555 560 Gly Gly Ser Gly Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu                 565 570 575 Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro             580 585 590 Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro         595 600 605 Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg     610 615 620 Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu 625 630 635 640 Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu                 645 650 655 Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser             660 665 670 His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr         675 680 685 Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro     690 695 700 Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr 705 710 715 720 Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp                 725 730 735 Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser             740 745 750 Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys         755 760 765

Claims (16)

a. A first peptide comprising an extracellular ligand binding domain of a VEGF receptor,
b. The Fc region of the antibody, and
c. A second peptide comprising an extracellular ligand binding domain of a PDGF receptor,
Wherein said fusion protein is arranged at the N-terminus to the C-terminus in an order selected from the group consisting of (a) - (b) - (c) and (c) - (b) - (a);
Wherein said fusion protein is capable of binding to VEGF and PDGF, and capable of inhibiting the activity of VEGF and the activity of PDGF.
The method according to claim 1,
a. The extracellular ligand binding domain of the VEGF receptor comprises an Ig-like domain D2 of VEGFRl and an Ig-like domain D3 of VEGFR2;
b. The Fc region of the antibody comprises the CH2 and CH3 regions of IgG1;
c. Wherein the extracellular ligand binding domain of the PDGF receptor comprises Ig-like domains D1 to D3 of PDGFR [beta].
The method of claim 2,
a. The extracellular ligand binding domain of the VEGF receptor comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 7;
b. The Fc region of the antibody comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 12;
c. Wherein the extracellular ligand binding domain of the PDGF receptor comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 2.
The method of claim 3,
a. The extracellular ligand binding domain of the VEGF receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 10;
b. The Fc region of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 15;
c. Wherein the extracellular ligand binding domain of the PDGF receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 5.
5. The method according to any one of claims 1 to 4,
Further comprising a first linker peptide between the Fc region and the first or second peptide at the C-terminus of the fusion protein and optionally a second linker peptide between the second or first peptide at the N-terminus of the fusion protein And a second linker peptide between the Fc regions.
The method of claim 5,
Wherein the first linker peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 30 and 32, and the second linker peptide comprises the amino acid sequence SEQ ID NO: Fusion protein.
The method according to any one of claims 1 to 6,
Wherein the fusion protein further comprises a signal peptide linked to the N-terminus of the fusion protein.
A fusion protein comprising an amino acid sequence having at least 90% homology with the 20-766 amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, the 21-769 amino acid sequence of SEQ ID NO: 44, or the 20-768 amino acid sequence of SEQ ID NO: .
The method of claim 8,
The amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 40, 20-766 amino acids of SEQ ID NO: 42, 21-769 amino acids of SEQ ID NO: 44 and 20-768 amino acids of SEQ ID NO: 50.
An isolated nucleic acid molecule encoding said fusion protein of any one of claims 1 to 9.
A host cell comprising a nucleic acid molecule encoding the fusion protein of any one of claims 1 to 9.
(1) culturing a host cell comprising a nucleic acid molecule encoding the fusion protein of any one of claims 1 to 9 under conditions in which the fusion protein is produced; And
(2) recovering the fusion protein produced by the host cell; and (3) a method for producing the fusion protein according to any one of claims 1 to 9.
A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 9 and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising a nucleic acid molecule encoding the fusion protein of any one of claims 1 to 9 and a pharmaceutically acceptable carrier.
Comprising the administration of an effective amount of the fusion protein of any one of claims 1 to 9 to an individual in need thereof, which comprises neovascularization, vascular permeability, edema and inflammation. A method of treating or preventing a clinical condition selected from the group.
Choroidal neovascularization, wet age-related macular degeneration, and map-shaped atrophy (see, for example, &lt; RTI ID = 0.0 &gt; geographic atrophy). &lt; / RTI &gt;

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